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Jinx CD8 + T cells produce a normal amount of IFN-γ upon polyclonal stimulation with <t>PMA/ionomycin</t> but fail to degranulate. (A) Surface expression of CD107a. Inset numbers indicate percentage of cells with induced expression. (B) Up-regulation of intracellular IFN-γ. Graphs beneath each FACS illustration show data for three mice.
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1) Product Images from "Jinx, an MCMV susceptibility phenotype caused by disruption of Unc13d: a mouse model of type 3 familial hemophagocytic lymphohistiocytosis"

Article Title: Jinx, an MCMV susceptibility phenotype caused by disruption of Unc13d: a mouse model of type 3 familial hemophagocytic lymphohistiocytosis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20062447

Jinx CD8 + T cells produce a normal amount of IFN-γ upon polyclonal stimulation with PMA/ionomycin but fail to degranulate. (A) Surface expression of CD107a. Inset numbers indicate percentage of cells with induced expression. (B) Up-regulation of intracellular IFN-γ. Graphs beneath each FACS illustration show data for three mice.
Figure Legend Snippet: Jinx CD8 + T cells produce a normal amount of IFN-γ upon polyclonal stimulation with PMA/ionomycin but fail to degranulate. (A) Surface expression of CD107a. Inset numbers indicate percentage of cells with induced expression. (B) Up-regulation of intracellular IFN-γ. Graphs beneath each FACS illustration show data for three mice.

Techniques Used: Expressing, FACS, Mouse Assay

2) Product Images from "Chemokine Receptor Expression Identifies Pre-T Helper (Th)1, Pre-Th2, and Nonpolarized Cells among Human CD4+ Central Memory T Cells"

Article Title: Chemokine Receptor Expression Identifies Pre-T Helper (Th)1, Pre-Th2, and Nonpolarized Cells among Human CD4+ Central Memory T Cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20040774

Ex vivo cytokine-producing capacities of CD4 + memory T cell subsets. (A) Purified CD4 + T CM and T EM cell subsets were stimulated with PdBu and ionomycin for 24 h and supernatants were analyzed for IFN-γ (diluted 1:4, white bars), IL-4 (black bars), and IL-5 (gray bars) by ELISA. Stimulation with anti-CD3 and anti-CD28 antibodies gave similar results (not depicted). Shown is the mean of four experiments with cells from different donors. (B) CXCR3 + CD4 + T cells were sorted for CCR7 and CD62L expression as indicated and IFN-γ production was assessed as described above. The mean of three independent experiments with three different donors is shown.
Figure Legend Snippet: Ex vivo cytokine-producing capacities of CD4 + memory T cell subsets. (A) Purified CD4 + T CM and T EM cell subsets were stimulated with PdBu and ionomycin for 24 h and supernatants were analyzed for IFN-γ (diluted 1:4, white bars), IL-4 (black bars), and IL-5 (gray bars) by ELISA. Stimulation with anti-CD3 and anti-CD28 antibodies gave similar results (not depicted). Shown is the mean of four experiments with cells from different donors. (B) CXCR3 + CD4 + T cells were sorted for CCR7 and CD62L expression as indicated and IFN-γ production was assessed as described above. The mean of three independent experiments with three different donors is shown.

Techniques Used: Ex Vivo, Purification, Enzyme-linked Immunosorbent Assay, Expressing

Effects of polarizing cytokines on TCR- and cytokine-induced differentiation. Purified CFSE-labeled CD4 + T cell subsets were stimulated with DC plus TSST or IL-7 plus IL-15 in the absence or presence of IL-12 and neutralizing anti–IL-4 antibody (Th1-condition) or IL-4 and neutralizing anti–IL-12 antibody (Th2-condition). After 7 d, cells were stimulated with PdBu and ionomycin and cells of the same division number were analyzed for IFN-γ and IL-4 production by intracellular staining. The percentages of INF-γ + cells (white bars), IL-4 + cells (black bars), and of cells producing both cytokines (gray bars) are represented. One representative experiment out of five with different donors is shown.
Figure Legend Snippet: Effects of polarizing cytokines on TCR- and cytokine-induced differentiation. Purified CFSE-labeled CD4 + T cell subsets were stimulated with DC plus TSST or IL-7 plus IL-15 in the absence or presence of IL-12 and neutralizing anti–IL-4 antibody (Th1-condition) or IL-4 and neutralizing anti–IL-12 antibody (Th2-condition). After 7 d, cells were stimulated with PdBu and ionomycin and cells of the same division number were analyzed for IFN-γ and IL-4 production by intracellular staining. The percentages of INF-γ + cells (white bars), IL-4 + cells (black bars), and of cells producing both cytokines (gray bars) are represented. One representative experiment out of five with different donors is shown.

Techniques Used: Purification, Labeling, Staining

(A) Differentiation of T CM cell subsets in response to TCR or cytokine stimulation. Purified CD4 + T cell subsets were CFSE labeled and stimulated with TSST-loaded DCs or with IL-7 and IL-15 in the presence of neutralizing anti–IL-4 and anti–IL-12 antibodies. After 7 d, cells were stimulated with PdBu and ionomycin, stained with APC-labeled anti–IFN-γ and PE-labeled anti–IL-4 antibodies, and cells of the same division number were analyzed by flow cytometry. Unstimulated T cell subsets were also analyzed ex vivo as control. Numbers indicate the percentage of cells producing IFN-γ or IL-4. One respresentative experiment out of five is shown. (B and C) Modulation of homing receptor expression by cytokine-stimulated CD4 + memory T cell subsets. Purified CFSE-labeled CD4 + T cell subsets were stimulated with IL-7 and IL-15. T CM and T EM cells in B were sorted for CXCR5, CCR7, and CD62L expression, whereas in C they were sorted for CXCR5, CCR7, CXCR3, and CCR4 expression as indicated. After 7 d, cells in division four were analyzed for the expression of CXCR5, CCR7, and CD62L (B), or CXCR3, CCR4, and CCR5 (C). One representative donor out of four is shown.
Figure Legend Snippet: (A) Differentiation of T CM cell subsets in response to TCR or cytokine stimulation. Purified CD4 + T cell subsets were CFSE labeled and stimulated with TSST-loaded DCs or with IL-7 and IL-15 in the presence of neutralizing anti–IL-4 and anti–IL-12 antibodies. After 7 d, cells were stimulated with PdBu and ionomycin, stained with APC-labeled anti–IFN-γ and PE-labeled anti–IL-4 antibodies, and cells of the same division number were analyzed by flow cytometry. Unstimulated T cell subsets were also analyzed ex vivo as control. Numbers indicate the percentage of cells producing IFN-γ or IL-4. One respresentative experiment out of five is shown. (B and C) Modulation of homing receptor expression by cytokine-stimulated CD4 + memory T cell subsets. Purified CFSE-labeled CD4 + T cell subsets were stimulated with IL-7 and IL-15. T CM and T EM cells in B were sorted for CXCR5, CCR7, and CD62L expression, whereas in C they were sorted for CXCR5, CCR7, CXCR3, and CCR4 expression as indicated. After 7 d, cells in division four were analyzed for the expression of CXCR5, CCR7, and CD62L (B), or CXCR3, CCR4, and CCR5 (C). One representative donor out of four is shown.

Techniques Used: Purification, Labeling, Staining, Flow Cytometry, Cytometry, Ex Vivo, Expressing

(A) Kinetics and requirements of TCR- and cytokine-induced IFN-γ production. CFSE-labeled CD4 + memory T cells were stimulated for the indicated times with either anti-CD3 and anti-CD28 antibodies (squares), or with TNF-α, IL-12, and IL-18 in the absence (circles) or presence (triangles) of IL-7 and IL-15. IFN-γ production was analyzed by intracellular staining. Empty symbols indicate conditions with undivided cells, whereas filled symbols indicate conditions with dividing cells. The mean percentage of IFN-γ + cells of three independent experiments is plotted. (B) Cytokine-stimulated CXCR3 + T CM cells lacking IFN-γ–producing capacity become Th1 cell effector cells. Purified CFSE-labeled CXCR3 + T CM and CCR4 + T CM cells were stimulated with IL-7, IL-15, TNF-α, IL-12, and IL-18 for 60 h, and IFN-γ–secreting cells were purified by cell sorting. IFN-γ + and IFN-γ − cells were then expanded for an additional 5 d with IL-7 and IL-15, briefly stimulated with PdBu and ionomycin, and analyzed for IL-4 and IFN-γ production by intracellular staining. One representative donor out of three is shown.
Figure Legend Snippet: (A) Kinetics and requirements of TCR- and cytokine-induced IFN-γ production. CFSE-labeled CD4 + memory T cells were stimulated for the indicated times with either anti-CD3 and anti-CD28 antibodies (squares), or with TNF-α, IL-12, and IL-18 in the absence (circles) or presence (triangles) of IL-7 and IL-15. IFN-γ production was analyzed by intracellular staining. Empty symbols indicate conditions with undivided cells, whereas filled symbols indicate conditions with dividing cells. The mean percentage of IFN-γ + cells of three independent experiments is plotted. (B) Cytokine-stimulated CXCR3 + T CM cells lacking IFN-γ–producing capacity become Th1 cell effector cells. Purified CFSE-labeled CXCR3 + T CM and CCR4 + T CM cells were stimulated with IL-7, IL-15, TNF-α, IL-12, and IL-18 for 60 h, and IFN-γ–secreting cells were purified by cell sorting. IFN-γ + and IFN-γ − cells were then expanded for an additional 5 d with IL-7 and IL-15, briefly stimulated with PdBu and ionomycin, and analyzed for IL-4 and IFN-γ production by intracellular staining. One representative donor out of three is shown.

Techniques Used: Labeling, Staining, Purification, FACS

3) Product Images from "T Cell and Dendritic Cell Abnormalities Synergize to Expand Pro-Inflammatory T Cell Subsets Leading to Fatal Autoimmunity in B6.NZBc1 Lupus-Prone Mice"

Article Title: T Cell and Dendritic Cell Abnormalities Synergize to Expand Pro-Inflammatory T Cell Subsets Leading to Fatal Autoimmunity in B6.NZBc1 Lupus-Prone Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0075166

Myeloid DC from c1(88-100) and c1(70-100) mice demonstrate altered function and an enhanced ability to induce differentiation of Th1 cells. BMDC from 8–12 wk-old mice were expanded with FLT3L for 7 days and then co-cultured with OVA peptide and purified naïve CD4 + T cells from OT-II TCR Tg mice. On day 4, the cells were re-stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop or GolgiPlug, and analyzed by flow cytometry for cell surface DC (CD11c, CD11b, B220, MHC-II, B7.2) or T cell (CD3, CD4) markers and intracellular cytokine levels. (A) Scatterplots showing the percentage of IL-21-, IL-17- and IFN-γ-producing T cells. Results are clustered in groups based on the strain of T cells (top of the figure) with the DC strain shown at the bottom of the figure. Scatterplots showing the percentage of CD11c + CD11b + B220 − mDC (B) and CD11c + CD11b - B220 + pDC (C) expressing elevated levels of MHCII and B7.2, or IL-6 and IL-12. Results with the different strains of T cells have been pooled as no differences were noted between strains. Horizontal lines indicate the mean. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with *p
Figure Legend Snippet: Myeloid DC from c1(88-100) and c1(70-100) mice demonstrate altered function and an enhanced ability to induce differentiation of Th1 cells. BMDC from 8–12 wk-old mice were expanded with FLT3L for 7 days and then co-cultured with OVA peptide and purified naïve CD4 + T cells from OT-II TCR Tg mice. On day 4, the cells were re-stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop or GolgiPlug, and analyzed by flow cytometry for cell surface DC (CD11c, CD11b, B220, MHC-II, B7.2) or T cell (CD3, CD4) markers and intracellular cytokine levels. (A) Scatterplots showing the percentage of IL-21-, IL-17- and IFN-γ-producing T cells. Results are clustered in groups based on the strain of T cells (top of the figure) with the DC strain shown at the bottom of the figure. Scatterplots showing the percentage of CD11c + CD11b + B220 − mDC (B) and CD11c + CD11b - B220 + pDC (C) expressing elevated levels of MHCII and B7.2, or IL-6 and IL-12. Results with the different strains of T cells have been pooled as no differences were noted between strains. Horizontal lines indicate the mean. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with *p

Techniques Used: Mouse Assay, Cell Culture, Purification, Flow Cytometry, Cytometry, Expressing

Expansion of Tfh, Th17 and Th1 cell subsets in c1 congenic mice. (A) Splenocytes from 4-mo-old mice were stained to assess the proportion of Tfh (CD4 + CD44 hi CD62L lo CXCR5 hi PD1 hi ) cells. Representative contour plots from B6 and c1(70-100) mice. Thick boxes denote the regions that were used to identify Tfh cells. Cells shown in the right panels were gated on the regions shown in the left panels. (B) Scatter plots showing the proportion of Tfh cells within the CD4 + T cell subset and absolute number of splenic Tfh cells. (C) Representative contour plots and histograms from flow cytometry analysis of IL-17-, IFN-γ-, and IL-4-expressing CD4 + T cells in B6 and c1(70-100) mice. Splenocytes were stimulated with PMA and ionomycin in the presence of GolgiStop for 4 h, and then fixed, stained with anti-CD3 and -CD4, permeabilized, and stained with anti-cytokine Ab. Thick lines outline the regions used to gate CD4 + CD3 + T cells. For histograms, the percentage of cells staining positively for each cytokine is indicated. (D) Scatterplots showing the percentages of cytokine-producing cells as a proportion of the CD4 + T cell population. Horizontal lines indicate the mean of each group examined. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with **p
Figure Legend Snippet: Expansion of Tfh, Th17 and Th1 cell subsets in c1 congenic mice. (A) Splenocytes from 4-mo-old mice were stained to assess the proportion of Tfh (CD4 + CD44 hi CD62L lo CXCR5 hi PD1 hi ) cells. Representative contour plots from B6 and c1(70-100) mice. Thick boxes denote the regions that were used to identify Tfh cells. Cells shown in the right panels were gated on the regions shown in the left panels. (B) Scatter plots showing the proportion of Tfh cells within the CD4 + T cell subset and absolute number of splenic Tfh cells. (C) Representative contour plots and histograms from flow cytometry analysis of IL-17-, IFN-γ-, and IL-4-expressing CD4 + T cells in B6 and c1(70-100) mice. Splenocytes were stimulated with PMA and ionomycin in the presence of GolgiStop for 4 h, and then fixed, stained with anti-CD3 and -CD4, permeabilized, and stained with anti-cytokine Ab. Thick lines outline the regions used to gate CD4 + CD3 + T cells. For histograms, the percentage of cells staining positively for each cytokine is indicated. (D) Scatterplots showing the percentages of cytokine-producing cells as a proportion of the CD4 + T cell population. Horizontal lines indicate the mean of each group examined. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with **p

Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing

4) Product Images from "CD11c+ monocyte/macrophages promote chronic Helicobacter hepaticus-induced intestinal inflammation through the production of IL-23"

Article Title: CD11c+ monocyte/macrophages promote chronic Helicobacter hepaticus-induced intestinal inflammation through the production of IL-23

Journal: Mucosal Immunology

doi: 10.1038/mi.2015.65

MHCII + monocytes and pathogenic T cells are reduced in Helicobacter hepaticus ( Hh )-infected and anti-IL-10R-treated CD11c IL-23- mice. CD11c IL-23+ and CD11c IL-23- mice were infected with Hh combined with anti-IL-10R monoclonal antibody (mAb) treatment and analyzed after 3 weeks. ( a ) Representative fluorescence-activated cell sorting (FACS) plots, frequencies among CD45 + leukocytes, and absolute numbers of the indicated myeloid subsets in the colonic lamina propria. ( b ) Frequencies of CD4 + T cells among CD45 + leukocytes. ( c ) Representative FACS plots and frequencies of IFNγ + , IFNγ + IL-17A + , and IL-17A + cells among CD4 + T cells upon restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Data points represent individual mice, bars indicate medians. Data are representative of two independent experiments. * P
Figure Legend Snippet: MHCII + monocytes and pathogenic T cells are reduced in Helicobacter hepaticus ( Hh )-infected and anti-IL-10R-treated CD11c IL-23- mice. CD11c IL-23+ and CD11c IL-23- mice were infected with Hh combined with anti-IL-10R monoclonal antibody (mAb) treatment and analyzed after 3 weeks. ( a ) Representative fluorescence-activated cell sorting (FACS) plots, frequencies among CD45 + leukocytes, and absolute numbers of the indicated myeloid subsets in the colonic lamina propria. ( b ) Frequencies of CD4 + T cells among CD45 + leukocytes. ( c ) Representative FACS plots and frequencies of IFNγ + , IFNγ + IL-17A + , and IL-17A + cells among CD4 + T cells upon restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Data points represent individual mice, bars indicate medians. Data are representative of two independent experiments. * P

Techniques Used: Infection, Mouse Assay, Fluorescence, FACS

5) Product Images from "T cell-specific inhibition of multiple apoptotic pathways blocks negative selection and causes autoimmunity"

Article Title: T cell-specific inhibition of multiple apoptotic pathways blocks negative selection and causes autoimmunity

Journal: eLife

doi: 10.7554/eLife.03468

T cell autoimmune pathology is apparent in BH3 Tg mice. ( A ) Quantification of flow cytometric analysis of T helper cell cytokine expression. Splenic CD4 T cells from 50-week-old mice were stimulated with PMA and Ionomycin for 4 hr in the presence of brefeldin A to allow cytokine accumulation. n ≥ 3 mice per genotype. ( B ) Hematoxylin and Eosin staining for lymphocyte infiltrates in liver, lung and kidney sections from 50-week-old mice. Images were captured at 20 x magnification. Data are representative of at least three mice per genotype. ( C ) Sera immunoblots for detection of autoantibodies. Whole tissue extracts were probed with sera from 40 to 50-week-old mice. Each lane corresponds to sera from an individual mouse: WT n = 2, BH3 Tg n = 7, Bcl-2 Tg n = 6. ( D ) Serum anti-nuclear antibody (ANA) quantification by ELISA. Dashed line represents two standard deviations above the WT mean. Samples above this line are considered positive for ANA. ( E ) Quantification of CD4 + Foxp3 + T reg cells in the lymphoid organs of aged mice. ( F ) Percentage of CD4 T cells that are T reg cells (CD25 + vs CD25 − ) in the mesenteric lymph nodes of 45 to 60-week-old mice. n ≥ 5 mice per genotype. DOI: http://dx.doi.org/10.7554/eLife.03468.012
Figure Legend Snippet: T cell autoimmune pathology is apparent in BH3 Tg mice. ( A ) Quantification of flow cytometric analysis of T helper cell cytokine expression. Splenic CD4 T cells from 50-week-old mice were stimulated with PMA and Ionomycin for 4 hr in the presence of brefeldin A to allow cytokine accumulation. n ≥ 3 mice per genotype. ( B ) Hematoxylin and Eosin staining for lymphocyte infiltrates in liver, lung and kidney sections from 50-week-old mice. Images were captured at 20 x magnification. Data are representative of at least three mice per genotype. ( C ) Sera immunoblots for detection of autoantibodies. Whole tissue extracts were probed with sera from 40 to 50-week-old mice. Each lane corresponds to sera from an individual mouse: WT n = 2, BH3 Tg n = 7, Bcl-2 Tg n = 6. ( D ) Serum anti-nuclear antibody (ANA) quantification by ELISA. Dashed line represents two standard deviations above the WT mean. Samples above this line are considered positive for ANA. ( E ) Quantification of CD4 + Foxp3 + T reg cells in the lymphoid organs of aged mice. ( F ) Percentage of CD4 T cells that are T reg cells (CD25 + vs CD25 − ) in the mesenteric lymph nodes of 45 to 60-week-old mice. n ≥ 5 mice per genotype. DOI: http://dx.doi.org/10.7554/eLife.03468.012

Techniques Used: Mouse Assay, Flow Cytometry, Expressing, Staining, Western Blot, Enzyme-linked Immunosorbent Assay

6) Product Images from "CXCL4 is a novel inducer of human Th17 cells and correlates with IL‐17 and IL‐22 in psoriatic arthritis"

Article Title: CXCL4 is a novel inducer of human Th17 cells and correlates with IL‐17 and IL‐22 in psoriatic arthritis

Journal: European Journal of Immunology

doi: 10.1002/eji.201747195

CXCL4 induces IL‐17 production in autologous antigen‐presenting cells (APCs)‐CD4 + T cells co‐culture. Monocytes, B cells, myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), and CD4 + T cells were isolated from healthy individuals, co‐cultured in the absence or presence of superantigen from Staphylococcal enterotoxin B (SEB) and CXCL4 for three days and restimulated with PMA and ionomycin. (A) Supernatant from co‐culture of monocytes and CD4 + T cells stimulated with superantigen SEB and CXCL4 were measured for IL‐17, IL‐22, IFN‐γ, and IL‐5. (B) The effect of CXCL4 treatment on 100 pg/mL superantigen SEB‐activated CD4 + T cells co‐cultured with myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), or B cells, on IL‐17, IL‐22, IFN‐γ, and IL‐5 production was assessed. Cytokines produced were determined using a Luminex‐based assay. Means (bars) and values from each donor are shown. Data are pooled from two to five independent experiments, with one to four donor samples in duplicate per experiment. Each dot on the bar graphs represent a single donor paired t ‐test was used for statistical analysis. * p
Figure Legend Snippet: CXCL4 induces IL‐17 production in autologous antigen‐presenting cells (APCs)‐CD4 + T cells co‐culture. Monocytes, B cells, myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), and CD4 + T cells were isolated from healthy individuals, co‐cultured in the absence or presence of superantigen from Staphylococcal enterotoxin B (SEB) and CXCL4 for three days and restimulated with PMA and ionomycin. (A) Supernatant from co‐culture of monocytes and CD4 + T cells stimulated with superantigen SEB and CXCL4 were measured for IL‐17, IL‐22, IFN‐γ, and IL‐5. (B) The effect of CXCL4 treatment on 100 pg/mL superantigen SEB‐activated CD4 + T cells co‐cultured with myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), or B cells, on IL‐17, IL‐22, IFN‐γ, and IL‐5 production was assessed. Cytokines produced were determined using a Luminex‐based assay. Means (bars) and values from each donor are shown. Data are pooled from two to five independent experiments, with one to four donor samples in duplicate per experiment. Each dot on the bar graphs represent a single donor paired t ‐test was used for statistical analysis. * p

Techniques Used: Co-Culture Assay, Isolation, Cell Culture, Produced, Luminex

CXCL4‐differentiated monocyte‐derived dendritic cells enhance pro‐inflammatory cytokine production and proliferation by CD4 + T cells. Monocytes from healthy donors were isolated and differentiated into dendritic cells in the absence or presence of CXCL4 (moDCs or CXCL4‐moDCs). moDCs were then co‐cultured with autologous CD4 + T cells in the presence of superantigen from Staphylococcal Enterotoxin B (SEB) for three days and restimulated with PMA and ionomycin. (A‐C) Comparison of co‐culture with moDC or CXCL4‐moDC on IL‐17 or IFN‐γ production by CD4 + T cells was assessed by (A, C) intracellular cytokine staining and (B) enzyme‐linked immunosorbent assay are shown. (D) Intracellular cytokine staining was performed for the measurement of IL‐4 + , IL‐10 + , IL‐22 + , and IL‐17 + IFN‐γ + cells gated on live CD4 + T cells. (E) The amount of IL‐17 producing cells co‐expressing IL‐22 as measured by flow cytometry. (F) CD4 + T cells were labeled with CellTrace Violet prior co‐culture and proliferation was analyzed as division index. Cells were gated on live, single, CD4 + T cells. Means (bars) and values from each donor are shown. Data are pooled from two to three independent experiments, with two to three donor samples per experiment. Each dot on the bar graphs represent a single donor and paired t ‐test was used for statistical analysis. * p
Figure Legend Snippet: CXCL4‐differentiated monocyte‐derived dendritic cells enhance pro‐inflammatory cytokine production and proliferation by CD4 + T cells. Monocytes from healthy donors were isolated and differentiated into dendritic cells in the absence or presence of CXCL4 (moDCs or CXCL4‐moDCs). moDCs were then co‐cultured with autologous CD4 + T cells in the presence of superantigen from Staphylococcal Enterotoxin B (SEB) for three days and restimulated with PMA and ionomycin. (A‐C) Comparison of co‐culture with moDC or CXCL4‐moDC on IL‐17 or IFN‐γ production by CD4 + T cells was assessed by (A, C) intracellular cytokine staining and (B) enzyme‐linked immunosorbent assay are shown. (D) Intracellular cytokine staining was performed for the measurement of IL‐4 + , IL‐10 + , IL‐22 + , and IL‐17 + IFN‐γ + cells gated on live CD4 + T cells. (E) The amount of IL‐17 producing cells co‐expressing IL‐22 as measured by flow cytometry. (F) CD4 + T cells were labeled with CellTrace Violet prior co‐culture and proliferation was analyzed as division index. Cells were gated on live, single, CD4 + T cells. Means (bars) and values from each donor are shown. Data are pooled from two to three independent experiments, with two to three donor samples per experiment. Each dot on the bar graphs represent a single donor and paired t ‐test was used for statistical analysis. * p

Techniques Used: Derivative Assay, Isolation, Cell Culture, Co-Culture Assay, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Cytometry, Labeling

7) Product Images from "Cytotoxic effector functions of T cells are not required for protective immunity against fatal Rickettsia typhi infection in a murine model of infection: Role of TH1 and TH17 cytokines in protection and pathology"

Article Title: Cytotoxic effector functions of T cells are not required for protective immunity against fatal Rickettsia typhi infection in a murine model of infection: Role of TH1 and TH17 cytokines in protection and pathology

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0005404

BALB/c mice generate cytotoxic CD8 + cells that are sporadically reactivated. BALB/c mice were infected with 1×10 6 sfu R . typhi . Control mice received PBS instead and were used as
Figure Legend Snippet: BALB/c mice generate cytotoxic CD8 + cells that are sporadically reactivated. BALB/c mice were infected with 1×10 6 sfu R . typhi . Control mice received PBS instead and were used as "day 0" control. Spleen cells were isolated and stained for CD8, KLRG1 and CD11a or restimulated with PMA/Ionomycin for 4h and stained for CD8 and intracellular IFNγ and Granzyme B. The dot plots show example stainings from day 7 post infection. Mice were analyzed for cytokine and Granzyme B expression on day 0, 7 and 15 (n = 6) and day 35 (n = 4). 3–4 mice were analyzed for KLRG1 and CD11a expression. Graphs show the percentage of KLRG1 + , CD11a + , Granzyme B + and IFNγ + T cells among CD8 + T cells (y-axis) at indicated days post infection (x-axis). Graphs show combined results from 2 independent experiments. Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test). Asterisks indicate significant differences compared to day 0 (* p

Techniques Used: Mouse Assay, Infection, Isolation, Staining, Expressing

BALB/c mice generate CD4 + T H 1 cells that are sporadically reactivated. Spleen cells from the same mice as described in Fig 1 were stained for CD4, CD11a and for intracellular IFNγ and Granzyme B after PMA/Ionomycin restimulation. The dot plots show example stainings from day 7 or day 15 post infection. Graphs show the percentage of CD11a + , IFNγ + and Granzyme B + T cells among CD4 + T cells (y-axis) at indicated days post infection (x-axis). Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test). Asterisks indicate significant differences compared to day 0 (* p
Figure Legend Snippet: BALB/c mice generate CD4 + T H 1 cells that are sporadically reactivated. Spleen cells from the same mice as described in Fig 1 were stained for CD4, CD11a and for intracellular IFNγ and Granzyme B after PMA/Ionomycin restimulation. The dot plots show example stainings from day 7 or day 15 post infection. Graphs show the percentage of CD11a + , IFNγ + and Granzyme B + T cells among CD4 + T cells (y-axis) at indicated days post infection (x-axis). Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test). Asterisks indicate significant differences compared to day 0 (* p

Techniques Used: Mouse Assay, Staining, Infection

8) Product Images from "Phenotypic Analysis of Prostate-Infiltrating Lymphocytes Reveals TH17 and Treg Skewing"

Article Title: Phenotypic Analysis of Prostate-Infiltrating Lymphocytes Reveals TH17 and Treg Skewing

Journal:

doi: 10.1158/1078-0432.CCR-07-5164

Frequency of T helper subsets in peripheral blood and prostate tissue of patients with prostate cancer. Positively isolated CD4 + T cells were stimulated for 4 h in the presence of phorbol 12-myristate13-acetate and ionomycin and analyzed by flow cytometry.
Figure Legend Snippet: Frequency of T helper subsets in peripheral blood and prostate tissue of patients with prostate cancer. Positively isolated CD4 + T cells were stimulated for 4 h in the presence of phorbol 12-myristate13-acetate and ionomycin and analyzed by flow cytometry.

Techniques Used: Isolation, Flow Cytometry, Cytometry

9) Product Images from "Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4+ T Cell Pathogenicity and Suppresses Autoimmunity"

Article Title: Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4+ T Cell Pathogenicity and Suppresses Autoimmunity

Journal: Cell Metabolism

doi: 10.1016/j.cmet.2019.10.015

PKM2 Tetramerization Blocks T Cell Activation In Vitro Murine CD4 + CD62 + T cells were stimulated in vitro with CD3/CD28 antibodies in the presence of DMSO (CTRL), TEPP-46 50 μM, or 100 μM. (A and B) Cells were collected after 24 h of stimulation. (A) Quantification of Il2 mRNA in activated T cells by qRT-PCR (n = 9 from three independent experiments). (B) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. IL-2 production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced IL-2 production by TEPP-46-treated cells. Right, quantification of the percentage of IL-2-producing cells and IL-2 mean fluorescence intensity (MFI) in CTRL versus TEPP-46-treated cells (n = 8 from 3 independent experiments). (C–F) Cells were collected after 3 days of stimulation. (C) Top, representative flow cytometry plot displaying T cell proliferation assessed as CellTrace violet dilution. Bottom, a division index was calculated with FlowJo software to quantify T cell proliferation (n = 5 from four independent experiments). (D) Expression of surface CD62L, CD44, and CD25 was evaluated by flow cytometry. The percentage of expressing cells and the MFI are shown (n = 3 from 2 independent experiments). (E) Quantification of Tnfa mRNA levels in activated T cells by qRT-PCR (n = 6 from 6 independent experiments). (F) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. TNF-α production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of TNF-α-producing cells and TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 5 from 2 independent experiments). For all panels, data are the mean ± SD. ∗ p
Figure Legend Snippet: PKM2 Tetramerization Blocks T Cell Activation In Vitro Murine CD4 + CD62 + T cells were stimulated in vitro with CD3/CD28 antibodies in the presence of DMSO (CTRL), TEPP-46 50 μM, or 100 μM. (A and B) Cells were collected after 24 h of stimulation. (A) Quantification of Il2 mRNA in activated T cells by qRT-PCR (n = 9 from three independent experiments). (B) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. IL-2 production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced IL-2 production by TEPP-46-treated cells. Right, quantification of the percentage of IL-2-producing cells and IL-2 mean fluorescence intensity (MFI) in CTRL versus TEPP-46-treated cells (n = 8 from 3 independent experiments). (C–F) Cells were collected after 3 days of stimulation. (C) Top, representative flow cytometry plot displaying T cell proliferation assessed as CellTrace violet dilution. Bottom, a division index was calculated with FlowJo software to quantify T cell proliferation (n = 5 from four independent experiments). (D) Expression of surface CD62L, CD44, and CD25 was evaluated by flow cytometry. The percentage of expressing cells and the MFI are shown (n = 3 from 2 independent experiments). (E) Quantification of Tnfa mRNA levels in activated T cells by qRT-PCR (n = 6 from 6 independent experiments). (F) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. TNF-α production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of TNF-α-producing cells and TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 5 from 2 independent experiments). For all panels, data are the mean ± SD. ∗ p

Techniques Used: Activation Assay, In Vitro, Quantitative RT-PCR, Flow Cytometry, Staining, Fluorescence, Software, Expressing

10) Product Images from "Memory T cell–driven differentiation of naive cells impairs adoptive immunotherapy"

Article Title: Memory T cell–driven differentiation of naive cells impairs adoptive immunotherapy

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI81217

T Mem cause precocious differentiation of naive cells. ( A ) Representative FACS and ( B ) bar graph summarizing the distribution of Ly5.1 + CD8 + T N -derived cell subsets 6 days following priming with CD3/CD28-specific antibodies and IL-2 alone or with Ly5.2 + CD8 + T Mem cells. Data shown after gating on Ly5.1 + CD8 + cells. ( C ) qPCR analysis of Sell , Ccr7 , and Cd27 expression in FACS-sorted reisolated T N cells primed alone, T N cells primed with T Mem cells, or T Mem cells primed alone. ( D ) Granzyme B and ( E ) IFN-γ intracellular staining in T N -derived cells stimulated with PMA/ionomycin following expansion alone or with T Mem cells. ( F ) IFN-γ ELISA of supernatants from reisolated T N -derived cells expanded alone or with T Mem cells 6 days prior to overnight stimulation with hgp100 25–33 peptide. ( G ) Heat maps of differentially expressed genes (1-way ANOVA, pFDR
Figure Legend Snippet: T Mem cause precocious differentiation of naive cells. ( A ) Representative FACS and ( B ) bar graph summarizing the distribution of Ly5.1 + CD8 + T N -derived cell subsets 6 days following priming with CD3/CD28-specific antibodies and IL-2 alone or with Ly5.2 + CD8 + T Mem cells. Data shown after gating on Ly5.1 + CD8 + cells. ( C ) qPCR analysis of Sell , Ccr7 , and Cd27 expression in FACS-sorted reisolated T N cells primed alone, T N cells primed with T Mem cells, or T Mem cells primed alone. ( D ) Granzyme B and ( E ) IFN-γ intracellular staining in T N -derived cells stimulated with PMA/ionomycin following expansion alone or with T Mem cells. ( F ) IFN-γ ELISA of supernatants from reisolated T N -derived cells expanded alone or with T Mem cells 6 days prior to overnight stimulation with hgp100 25–33 peptide. ( G ) Heat maps of differentially expressed genes (1-way ANOVA, pFDR

Techniques Used: FACS, Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Staining, Enzyme-linked Immunosorbent Assay

11) Product Images from "Development Of Foxp3+ Regulatory T Cells Is Driven By A c-Rel Enhanceosome"

Article Title: Development Of Foxp3+ Regulatory T Cells Is Driven By A c-Rel Enhanceosome

Journal: Immunity

doi: 10.1016/j.immuni.2009.10.006

c-Rel mediates Treg differentiation independent of IL-2 (a) Purified CD4 + CD25 − T cells from 6-week-old WT and c-Rel −/− mice (n=5) were cultured in the presence of plate-bound anti-CD3, soluble anti-CD28, anti-IL-2 (1 μg/ml), anti-IL-4, anti-IFN-γ, and TGF-β for three days, re-stimulated with PMA and ionomycin for 4 hours, stained with antibodies to IL-17A and Foxp3, and analyzed by flow cytometry. (b) Percentages of CD4 + Foxp3 + cells after in vitro Treg differentiation as shown in panel a, in the presence or absence of anti-IL-2. Results are representative of three independent experiments. *, p
Figure Legend Snippet: c-Rel mediates Treg differentiation independent of IL-2 (a) Purified CD4 + CD25 − T cells from 6-week-old WT and c-Rel −/− mice (n=5) were cultured in the presence of plate-bound anti-CD3, soluble anti-CD28, anti-IL-2 (1 μg/ml), anti-IL-4, anti-IFN-γ, and TGF-β for three days, re-stimulated with PMA and ionomycin for 4 hours, stained with antibodies to IL-17A and Foxp3, and analyzed by flow cytometry. (b) Percentages of CD4 + Foxp3 + cells after in vitro Treg differentiation as shown in panel a, in the presence or absence of anti-IL-2. Results are representative of three independent experiments. *, p

Techniques Used: Purification, Mouse Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, In Vitro

c-Rel deficiency blocks Foxp3 gene expression and Treg differentiation, whereas c-Rel co-expression increases Foxp3 promoter activity (a) Reduced Foxp3 mRNA expression in c-Rel −/− splenic T cells. Splenic CD4 + T cells were purified from 6-week-old WT and c-Rel −/− mice (n=5) and stimulated with anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) for 6 hrs, or PMA (50 ng/ml) and ionomycin (1 μM) for 2 hrs. Total RNA was extracted and Foxp3 mRNA levels were determined by real-time RT-PCR. (b) Reduced Foxp3 mRNA expression in c-Rel −/− T cells during Treg differentiation. Purified CD4 + CD25 − splenic T cells were cultured in the presence of plate-bound anti-CD3 (5 μg/ml), soluble anti-CD28 (1 μg/ml), IL-2 (50 U/ml), anti-IL-4 (1 μg/ml), anti-IFN-γ (1 μg/ml), and TGF-β (5 ng/ml) for the indicated times. Total RNA was extracted and Foxp3 mRNA levels were determined by real-time RT-PCR. ( c–d) Reduced Treg differentiation of c-Rel −/− T cells. CD4 + CD25 − T cells were cultured as in panel b for three days, re-stimulated with PMA (50 ng/ml) and ionomycin (1 μM) for 4 hrs, stained with antibodies to IL-17A and Foxp3, and analyzed by flow cytometry. (e–h) Activation of the Foxp3 promoter by c-Rel and p65, but not p50 or RelB. EL4/LAF cells were transiently transfected with murine Foxp3 promoter luciferase constructs together with an expression vector for full-length c-Rel (e), p65 (f), p50 (g), or RelB (h), or the empty vector as indicated. After 24 hrs, cells were treated with or without PMA (50 ng/ml) and ionomycin (1 μM) for 5 hrs, and the luciferase activities measured. The promoter activity is presented as fold increase over cells transfected with empty vector but not treated with PMA and ionomycin. To normalize the transfection efficiency across samples, the Renilla luciferase expression vector pRLTK was used as an internal control. *, The differences between the two groups are statistically significant (p
Figure Legend Snippet: c-Rel deficiency blocks Foxp3 gene expression and Treg differentiation, whereas c-Rel co-expression increases Foxp3 promoter activity (a) Reduced Foxp3 mRNA expression in c-Rel −/− splenic T cells. Splenic CD4 + T cells were purified from 6-week-old WT and c-Rel −/− mice (n=5) and stimulated with anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) for 6 hrs, or PMA (50 ng/ml) and ionomycin (1 μM) for 2 hrs. Total RNA was extracted and Foxp3 mRNA levels were determined by real-time RT-PCR. (b) Reduced Foxp3 mRNA expression in c-Rel −/− T cells during Treg differentiation. Purified CD4 + CD25 − splenic T cells were cultured in the presence of plate-bound anti-CD3 (5 μg/ml), soluble anti-CD28 (1 μg/ml), IL-2 (50 U/ml), anti-IL-4 (1 μg/ml), anti-IFN-γ (1 μg/ml), and TGF-β (5 ng/ml) for the indicated times. Total RNA was extracted and Foxp3 mRNA levels were determined by real-time RT-PCR. ( c–d) Reduced Treg differentiation of c-Rel −/− T cells. CD4 + CD25 − T cells were cultured as in panel b for three days, re-stimulated with PMA (50 ng/ml) and ionomycin (1 μM) for 4 hrs, stained with antibodies to IL-17A and Foxp3, and analyzed by flow cytometry. (e–h) Activation of the Foxp3 promoter by c-Rel and p65, but not p50 or RelB. EL4/LAF cells were transiently transfected with murine Foxp3 promoter luciferase constructs together with an expression vector for full-length c-Rel (e), p65 (f), p50 (g), or RelB (h), or the empty vector as indicated. After 24 hrs, cells were treated with or without PMA (50 ng/ml) and ionomycin (1 μM) for 5 hrs, and the luciferase activities measured. The promoter activity is presented as fold increase over cells transfected with empty vector but not treated with PMA and ionomycin. To normalize the transfection efficiency across samples, the Renilla luciferase expression vector pRLTK was used as an internal control. *, The differences between the two groups are statistically significant (p

Techniques Used: Expressing, Activity Assay, Purification, Mouse Assay, Quantitative RT-PCR, Cell Culture, Staining, Flow Cytometry, Cytometry, Activation Assay, Transfection, Luciferase, Construct, Plasmid Preparation

12) Product Images from "Regulatory and T effector cells have overlapping low to high ranges in TCR affinities for self during demyelinating disease"

Article Title: Regulatory and T effector cells have overlapping low to high ranges in TCR affinities for self during demyelinating disease

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1501464

Production of cytokines by Tconv and Treg cells during EAE. (A) Representative flow plots showing IL-17A and IFNγ production by CNS Tconv cells. (B) Frequencies of single and double cytokine producing Tconv cells in various organs at peak EAE upon stimulation with PMA and ionomycin. Data are from at least 5 independent experiments. (C) Percent of Tregs producing IL-10 or IFNγ in the various organs in response to PMA/Ionomycin stimulation. Data are from three independent experiments, except for SPL which is from two experiments.
Figure Legend Snippet: Production of cytokines by Tconv and Treg cells during EAE. (A) Representative flow plots showing IL-17A and IFNγ production by CNS Tconv cells. (B) Frequencies of single and double cytokine producing Tconv cells in various organs at peak EAE upon stimulation with PMA and ionomycin. Data are from at least 5 independent experiments. (C) Percent of Tregs producing IL-10 or IFNγ in the various organs in response to PMA/Ionomycin stimulation. Data are from three independent experiments, except for SPL which is from two experiments.

Techniques Used: Flow Cytometry

13) Product Images from "The E3 ubiquitin ligase adaptor Ndfip1 regulates TH17 differentiation by limiting the production of pro-inflammatory cytokines 1"

Article Title: The E3 ubiquitin ligase adaptor Ndfip1 regulates TH17 differentiation by limiting the production of pro-inflammatory cytokines 1

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1102779

Eosinophils are significant producers of IL-6 in Ndfip1 −/− mice (A) The amount of IL-6 in the supernatants of splenocytes restimulated for 4.5–5 hours with PMA and ionomycin was measured by ELISA. Bar graph represents the mean + s.d. of triplicate samples from one mouse for each genotype. The results are representative of 3–4 mice per genotype. *p
Figure Legend Snippet: Eosinophils are significant producers of IL-6 in Ndfip1 −/− mice (A) The amount of IL-6 in the supernatants of splenocytes restimulated for 4.5–5 hours with PMA and ionomycin was measured by ELISA. Bar graph represents the mean + s.d. of triplicate samples from one mouse for each genotype. The results are representative of 3–4 mice per genotype. *p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

14) Product Images from "The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR"

Article Title: The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR

Journal: Nature immunology

doi: 10.1038/s41590-018-0137-8

Itch and WWP2 collaboratively regulate T H 2 differentiation. a , Frequency of splenic memory-like (CD44 hi CD62L − ) CD4 + T cells (left) and naive (CD25 − CD44 − CD62L hi ) CD4 + T cells (middle) from 6- to 7-week-old wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre and DKO mice (key; n = 8 per group), and flow cytometry of splenic naive CD4 + T cells from such mice, showing gating used (right). Numbers adjacent to outlined areas (right) indicate percent CD4 + CD25 − CD44 − CD62L hi cells. b , Gene-expression profiles (left) of splenic CD4 + T cells stimulated for 4h with PMA and ionomycin, showing genes expressed differentially (mean of normalized counts) in Wwp2 −/− , Itch f/f Cd4 -Cre or DKO cells (above plots) relative to their expression in wild-type cells, and expression of genes encoding selected cytokines (middle) or transcriptional regulators (right) in cells as at left (above plots), presented as log 2 reads per kilobase of exon per million mapped reads (RPKM) values. c , Frequency of CD4 + T cells with intracellular staining of IL-4 (left) or IFN-γ (right) among splenocytes obtained from mice as in a (key; n = 5–6 per group (IL-4) or n = 4–5 per group (IFN-γ)) and stimulated for 5h in vitro with PMA and ionomycin in the presence of the protein-transport inhibitor GolgiStop. d , ELISA of IL-4 (left) or IFN-γ (right) in supernatants of CD4 + T cells sorted from mice as in a (key; n = 3 per group) and stimulated for 48h in vitro with anti-CD3 plus anti-CD28. e , f , Immunoblot analysis of GATA-3 ( e ) and phosphorylated (p-) and total STAT6 ( e ) or STAT5 ( f ), as well as actin (loading control) (left margin), in lysates of CD4 + T cells sorted from mice as in a (above lanes) and left unstimulated (left) or stimulated for 16h with anti-CD3 plus anti-CD28 (right; α-CD3 + α-CD28); numbers below lanes indicate the ratio of phosphorylated protein to total protein. Each symbol ( a , c , d ) represents an individual mouse; small horizontal lines ( a ) indicate the mean (±s.d.). * P
Figure Legend Snippet: Itch and WWP2 collaboratively regulate T H 2 differentiation. a , Frequency of splenic memory-like (CD44 hi CD62L − ) CD4 + T cells (left) and naive (CD25 − CD44 − CD62L hi ) CD4 + T cells (middle) from 6- to 7-week-old wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre and DKO mice (key; n = 8 per group), and flow cytometry of splenic naive CD4 + T cells from such mice, showing gating used (right). Numbers adjacent to outlined areas (right) indicate percent CD4 + CD25 − CD44 − CD62L hi cells. b , Gene-expression profiles (left) of splenic CD4 + T cells stimulated for 4h with PMA and ionomycin, showing genes expressed differentially (mean of normalized counts) in Wwp2 −/− , Itch f/f Cd4 -Cre or DKO cells (above plots) relative to their expression in wild-type cells, and expression of genes encoding selected cytokines (middle) or transcriptional regulators (right) in cells as at left (above plots), presented as log 2 reads per kilobase of exon per million mapped reads (RPKM) values. c , Frequency of CD4 + T cells with intracellular staining of IL-4 (left) or IFN-γ (right) among splenocytes obtained from mice as in a (key; n = 5–6 per group (IL-4) or n = 4–5 per group (IFN-γ)) and stimulated for 5h in vitro with PMA and ionomycin in the presence of the protein-transport inhibitor GolgiStop. d , ELISA of IL-4 (left) or IFN-γ (right) in supernatants of CD4 + T cells sorted from mice as in a (key; n = 3 per group) and stimulated for 48h in vitro with anti-CD3 plus anti-CD28. e , f , Immunoblot analysis of GATA-3 ( e ) and phosphorylated (p-) and total STAT6 ( e ) or STAT5 ( f ), as well as actin (loading control) (left margin), in lysates of CD4 + T cells sorted from mice as in a (above lanes) and left unstimulated (left) or stimulated for 16h with anti-CD3 plus anti-CD28 (right; α-CD3 + α-CD28); numbers below lanes indicate the ratio of phosphorylated protein to total protein. Each symbol ( a , c , d ) represents an individual mouse; small horizontal lines ( a ) indicate the mean (±s.d.). * P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Staining, In Vitro, Enzyme-linked Immunosorbent Assay

15) Product Images from "Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7–dependent lupus"

Article Title: Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7–dependent lupus

Journal: JCI Insight

doi: 10.1172/jci.insight.124729

Reduced in vivo Th1 and Th17 responses in imiquimod-treated Padi2 –/– and Padi4 –/– mice. ( A and B ) Splenocytes from untreated and imiquimod-treated (IMQ-treated) FVB, Padi2 –/– , and Padi4 –/– mice were stimulated in vitro with PMA and ionomycin, and CD4 + T cell cytokine production was measured by flow cytometry to determine Th1 ( A ) and Th17 ( B ) responses. Box-and-whisker plots show median, lower and upper quartiles, and minimum and maximum % values and are representative of 2 independent experiments, each performed in 4-6 mice/group. * P
Figure Legend Snippet: Reduced in vivo Th1 and Th17 responses in imiquimod-treated Padi2 –/– and Padi4 –/– mice. ( A and B ) Splenocytes from untreated and imiquimod-treated (IMQ-treated) FVB, Padi2 –/– , and Padi4 –/– mice were stimulated in vitro with PMA and ionomycin, and CD4 + T cell cytokine production was measured by flow cytometry to determine Th1 ( A ) and Th17 ( B ) responses. Box-and-whisker plots show median, lower and upper quartiles, and minimum and maximum % values and are representative of 2 independent experiments, each performed in 4-6 mice/group. * P

Techniques Used: In Vivo, Mouse Assay, In Vitro, Flow Cytometry, Cytometry, Whisker Assay

16) Product Images from "Therapeutic Helminth Infection of Macaques with Idiopathic Chronic Diarrhea Alters the Inflammatory Signature and Mucosal Microbiota of the Colon"

Article Title: Therapeutic Helminth Infection of Macaques with Idiopathic Chronic Diarrhea Alters the Inflammatory Signature and Mucosal Microbiota of the Colon

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003000

Flow cytometric analysis of CD4 + T cells from colon biopsies following T. trichiura treatment. Pinch biopsies collected during colonoscopies pre-treatment (Pre-Tt) and 14 weeks post-treatment (Post-Tt) were compared with samples collected from healthy age-matched controls (Control). Lamina propria mononuclear cells (LPMCs) from biopsies were stimulated ex vivo with PMA and ionomycin for intracellular cytokine staining. ( A ) Box and whisker plot showing the percentage of IL-4+ cells among CD4+ LPMCs. ***p
Figure Legend Snippet: Flow cytometric analysis of CD4 + T cells from colon biopsies following T. trichiura treatment. Pinch biopsies collected during colonoscopies pre-treatment (Pre-Tt) and 14 weeks post-treatment (Post-Tt) were compared with samples collected from healthy age-matched controls (Control). Lamina propria mononuclear cells (LPMCs) from biopsies were stimulated ex vivo with PMA and ionomycin for intracellular cytokine staining. ( A ) Box and whisker plot showing the percentage of IL-4+ cells among CD4+ LPMCs. ***p

Techniques Used: Flow Cytometry, Ex Vivo, Staining, Whisker Assay

17) Product Images from "TLR7 induces anergy in human CD4+ T cells"

Article Title: TLR7 induces anergy in human CD4+ T cells

Journal: Nature immunology

doi: 10.1038/ni.3036

IMQ inhibits JNK and Jun activation after full CD4 + T cell stimulation. CD4 + T cells were stimulated for 2 hours with PMA and Ionomycin in the presence or absence of IMQ. a. Histograms show a representative example of the expression of phosphorylated
Figure Legend Snippet: IMQ inhibits JNK and Jun activation after full CD4 + T cell stimulation. CD4 + T cells were stimulated for 2 hours with PMA and Ionomycin in the presence or absence of IMQ. a. Histograms show a representative example of the expression of phosphorylated

Techniques Used: Activation Assay, Cell Stimulation, Expressing

Mechanism of TLR7-induced anergy. a . Plots represent calcium fluxes as measured by Indo-1AM ratio over time in CD4 + T cells stimulated with two concentrations of IMQ (upper), ionomycin (lower left) or ssRNA40-LyoVec™ (lower right). Arrows indicate
Figure Legend Snippet: Mechanism of TLR7-induced anergy. a . Plots represent calcium fluxes as measured by Indo-1AM ratio over time in CD4 + T cells stimulated with two concentrations of IMQ (upper), ionomycin (lower left) or ssRNA40-LyoVec™ (lower right). Arrows indicate

Techniques Used:

18) Product Images from "USP18 inhibits NF-?B and NFAT activation during Th17 differentiation by deubiquitinating the TAK1-TAB1 complex"

Article Title: USP18 inhibits NF-?B and NFAT activation during Th17 differentiation by deubiquitinating the TAK1-TAB1 complex

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20122327

USP18 interacts with the TAK1–TAB complex. (A) TAK1 was isolated by immunoprecipitation from cells stimulated with anti-CD3/CD28 for the indicated time points, followed by kinase assay using recombinant MKK6 as a substrate. The intensities of the indicated proteins were quantified and the ratio of pMEKK6/TAK1 was calculated. (B) 293T cells were transfected with the indicated plasmids. 24 h later, cells were lysed and immunoprecipitation was performed with anti-Flag. The immunoprecipitants were analyzed by immunoblot with the indicated antibodies. The expression levels of TAK1 in the lysates were analyzed by immunoblot with anti-HA. (C) Jurkat T cells were stimulated with PMA and ionomycin for the indicated time points, followed by immunoprecipitation by control IgG (Ig) or anti-USP18 (αUSP18). The immunoprecipitants were analyzed by immunoblot with antibodies against the indicated proteins. The expression levels of CARMA1, TAK1, TAB1, TAB2, and Actin were analyzed by immunoblot with antibodies against the indicated proteins. Data shown are representatives of three (A and B) and two (C) independent experiments. Graphs show mean ± SD, n = 3. *, P
Figure Legend Snippet: USP18 interacts with the TAK1–TAB complex. (A) TAK1 was isolated by immunoprecipitation from cells stimulated with anti-CD3/CD28 for the indicated time points, followed by kinase assay using recombinant MKK6 as a substrate. The intensities of the indicated proteins were quantified and the ratio of pMEKK6/TAK1 was calculated. (B) 293T cells were transfected with the indicated plasmids. 24 h later, cells were lysed and immunoprecipitation was performed with anti-Flag. The immunoprecipitants were analyzed by immunoblot with the indicated antibodies. The expression levels of TAK1 in the lysates were analyzed by immunoblot with anti-HA. (C) Jurkat T cells were stimulated with PMA and ionomycin for the indicated time points, followed by immunoprecipitation by control IgG (Ig) or anti-USP18 (αUSP18). The immunoprecipitants were analyzed by immunoblot with antibodies against the indicated proteins. The expression levels of CARMA1, TAK1, TAB1, TAB2, and Actin were analyzed by immunoblot with antibodies against the indicated proteins. Data shown are representatives of three (A and B) and two (C) independent experiments. Graphs show mean ± SD, n = 3. *, P

Techniques Used: Isolation, Immunoprecipitation, Kinase Assay, Recombinant, Transfection, Expressing

USP18 catalyzes deubiquitination of TAK1. (A) Naive CD4 + T cells from USP18KO were sorted and transfected with empty vector (Vec), Flag-tagged USP18 (WT), or USP18(C61S) (CS). The transfected cells were sorted and stimulated with PMA and ionomycin (PMA/ion) for 10 min, followed by immunoblot analysis with antibodies against the indicated proteins. (B) 293T cells were transfected with USP18 together with HA-tagged TAK1 and TAB1, or CARMA1. TAK1 complex and CARMA1 were isolated by immunoprecipitation with anti-HA, followed by immunoblot analysis with anti-ubiquitin (αUb). The expression levels of TAK1, TAB1, and CARMA1 in lysates were analyzed by immunoblot with anti-HA. (C) 293T cells were transfected with HA-TAK1 with or without Myc-TAB1. The HA–TAK1 complex was immunoprecipitated with anti-HA. Flag-USP18 was purified by immunoprecipitation with anti-Flag agarose followed by elution with Flag peptide from 293T cells transfected with Flag-USP18. The immunoprecipitants were incubated with or with out Flag-USP18 protein followed by immunoblot with anti-HA or anti-ubiquitin (αUb). (D) WT and USP18KO (KO) CD4 + T cells were stimulated with PMA/ion for the indicated time points. The cells were lysed and cell lysates were immunoprecipitated with anti-TAK1. The immunoprecipitants were analyzed with anti-ubiquitin or anti-TAK1. The expression levels of β-actin were analyzed by immunoblot. (E) Splenocytes from WT and USP18KO (KO) mice left untreated or stimulated with PMA and ionomycin for 5 h were stained with fluorescence-labeled anti-CD4, CD44, CD69, and CD62L, followed by flow cytometry analysis. The flow blots show one representative of WT and KO mice, respectively. Graphs show mean ± SD, n = 4. Data shown are representatives of three (A–C) and two (D and E) independent experiments.
Figure Legend Snippet: USP18 catalyzes deubiquitination of TAK1. (A) Naive CD4 + T cells from USP18KO were sorted and transfected with empty vector (Vec), Flag-tagged USP18 (WT), or USP18(C61S) (CS). The transfected cells were sorted and stimulated with PMA and ionomycin (PMA/ion) for 10 min, followed by immunoblot analysis with antibodies against the indicated proteins. (B) 293T cells were transfected with USP18 together with HA-tagged TAK1 and TAB1, or CARMA1. TAK1 complex and CARMA1 were isolated by immunoprecipitation with anti-HA, followed by immunoblot analysis with anti-ubiquitin (αUb). The expression levels of TAK1, TAB1, and CARMA1 in lysates were analyzed by immunoblot with anti-HA. (C) 293T cells were transfected with HA-TAK1 with or without Myc-TAB1. The HA–TAK1 complex was immunoprecipitated with anti-HA. Flag-USP18 was purified by immunoprecipitation with anti-Flag agarose followed by elution with Flag peptide from 293T cells transfected with Flag-USP18. The immunoprecipitants were incubated with or with out Flag-USP18 protein followed by immunoblot with anti-HA or anti-ubiquitin (αUb). (D) WT and USP18KO (KO) CD4 + T cells were stimulated with PMA/ion for the indicated time points. The cells were lysed and cell lysates were immunoprecipitated with anti-TAK1. The immunoprecipitants were analyzed with anti-ubiquitin or anti-TAK1. The expression levels of β-actin were analyzed by immunoblot. (E) Splenocytes from WT and USP18KO (KO) mice left untreated or stimulated with PMA and ionomycin for 5 h were stained with fluorescence-labeled anti-CD4, CD44, CD69, and CD62L, followed by flow cytometry analysis. The flow blots show one representative of WT and KO mice, respectively. Graphs show mean ± SD, n = 4. Data shown are representatives of three (A–C) and two (D and E) independent experiments.

Techniques Used: Transfection, Plasmid Preparation, Isolation, Immunoprecipitation, Expressing, Purification, Incubation, Mouse Assay, Staining, Fluorescence, Labeling, Flow Cytometry, Cytometry

USP18KO cells defects in Th17 generation in vitro. (A) Naive CD4 + T cells were sorted by flow cytometry (gated on CD4 + CD25 − CD44 low CD62L high ) and stimulated with anti-CD3 and APC from WT or mice lacking B7.1, B7.2, and B7h to generate effector or tolerant T cells. After 5 d of culture, cells were washed and stimulated with anti-CD3 for 5 h, followed by real-time PCR analysis. (B) CD4 + and CD8 + T cells, memory (gated on CD4 + CD25 − CD44 low CD62L high ), nT reg cells (CD4 + CD25 + CD44 − CD62L − ), and B220 + B cells were sorted by flow cytometry from splenocytes. BMDCs and BMDMs were differentiated from BM progenitor cells with GM-CSF or M-CSF. Th0, Th1, Th2, iT reg, and Th17 cells were prepared by culturing naive cells in these polarizing conditions for 5 d, followed by stimulation with anti-CD3 for 24 h, followed by real-time analysis or by PMA and ionomycin for 5 h, followed by intracellular cytokine staining (not depicted) to examine the differentiation efficiency. (C) WT and USP18KO (KO) naive CD4 + T cells were cultured under different polarizing conditions for 4 d. Cells were washed and stimulated with PMA plus ionomycin in the presence of Golgi stop for 5 h, followed by intracellular staining of the indicated antibodies. (D–F) Naive CD4 + T cells from WT or USP18KO (KO) mice were purified and stimulated with anti-CD3/CD28 for 4 d. Cells were washed and stimulated with PMA plus ionomycin for 5 h, followed by intracellular cytokine staining (left plots), with anti-CD3 for 24 h for ELISA (right graph; D), or with anti-CD3 for 4 h for real-time PCR analysis (E), or stained with anti-IFNGR1 and -IFNGR2 (F). (G) Naive CD4 + T cells from WT or USP18KO (KO) mice were purified and cultured under Th17 polarizing conditions (anti-CD3/CD28, TGF-β, and IL-6) for 4 d. Cells were stimulated with PMA and ionomycin for 5 h followed by flow cytometry analysis. (H) Naive CD4 + T cells from WT or USP18KO (KO) mice were differentiated under Th17 condition for 48 h. Cells were harvested and real-time PCR analysis was performed to determine the mRNA levels of the indicated cytokines. The level of the lower sample for each gene was set at 1 for comparison. Data are representative from two (A and B) or at least three independent experiments (C–H). Bar graphs show mean ± SD, n = 3. *, P
Figure Legend Snippet: USP18KO cells defects in Th17 generation in vitro. (A) Naive CD4 + T cells were sorted by flow cytometry (gated on CD4 + CD25 − CD44 low CD62L high ) and stimulated with anti-CD3 and APC from WT or mice lacking B7.1, B7.2, and B7h to generate effector or tolerant T cells. After 5 d of culture, cells were washed and stimulated with anti-CD3 for 5 h, followed by real-time PCR analysis. (B) CD4 + and CD8 + T cells, memory (gated on CD4 + CD25 − CD44 low CD62L high ), nT reg cells (CD4 + CD25 + CD44 − CD62L − ), and B220 + B cells were sorted by flow cytometry from splenocytes. BMDCs and BMDMs were differentiated from BM progenitor cells with GM-CSF or M-CSF. Th0, Th1, Th2, iT reg, and Th17 cells were prepared by culturing naive cells in these polarizing conditions for 5 d, followed by stimulation with anti-CD3 for 24 h, followed by real-time analysis or by PMA and ionomycin for 5 h, followed by intracellular cytokine staining (not depicted) to examine the differentiation efficiency. (C) WT and USP18KO (KO) naive CD4 + T cells were cultured under different polarizing conditions for 4 d. Cells were washed and stimulated with PMA plus ionomycin in the presence of Golgi stop for 5 h, followed by intracellular staining of the indicated antibodies. (D–F) Naive CD4 + T cells from WT or USP18KO (KO) mice were purified and stimulated with anti-CD3/CD28 for 4 d. Cells were washed and stimulated with PMA plus ionomycin for 5 h, followed by intracellular cytokine staining (left plots), with anti-CD3 for 24 h for ELISA (right graph; D), or with anti-CD3 for 4 h for real-time PCR analysis (E), or stained with anti-IFNGR1 and -IFNGR2 (F). (G) Naive CD4 + T cells from WT or USP18KO (KO) mice were purified and cultured under Th17 polarizing conditions (anti-CD3/CD28, TGF-β, and IL-6) for 4 d. Cells were stimulated with PMA and ionomycin for 5 h followed by flow cytometry analysis. (H) Naive CD4 + T cells from WT or USP18KO (KO) mice were differentiated under Th17 condition for 48 h. Cells were harvested and real-time PCR analysis was performed to determine the mRNA levels of the indicated cytokines. The level of the lower sample for each gene was set at 1 for comparison. Data are representative from two (A and B) or at least three independent experiments (C–H). Bar graphs show mean ± SD, n = 3. *, P

Techniques Used: In Vitro, Flow Cytometry, Cytometry, Mouse Assay, Real-time Polymerase Chain Reaction, Staining, Cell Culture, Purification, Enzyme-linked Immunosorbent Assay

19) Product Images from "Modulation of Innate and Adaptive Immune Responses by Tofacitinib (CP-690,550)"

Article Title: Modulation of Innate and Adaptive Immune Responses by Tofacitinib (CP-690,550)

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1003668

CP-690,550 inhibits the differentiation of IL-23-induced Th17 cells and associated cytokines. Sorted naïve CD4 + T cells were activated in serum-free media in the absence or presence of CP-690,550 with anti-CD3/anti-CD28 antibodies and the combination of IL-6, IL-1β and either TGF-β1 or IL-23. After 4 days of culture cells were restimulated with PMA/ionomycin and the expression of IL-17A, IL-17F and IL-22 was assessed by flow cytometry ( A ). Expression of Il21 ( B ) and Tbx21 ( C ) was determined by quantitative RT-PCR. Results were normalized using β-actin transcripts and represent relative expression (mean ± SEM, B , * P
Figure Legend Snippet: CP-690,550 inhibits the differentiation of IL-23-induced Th17 cells and associated cytokines. Sorted naïve CD4 + T cells were activated in serum-free media in the absence or presence of CP-690,550 with anti-CD3/anti-CD28 antibodies and the combination of IL-6, IL-1β and either TGF-β1 or IL-23. After 4 days of culture cells were restimulated with PMA/ionomycin and the expression of IL-17A, IL-17F and IL-22 was assessed by flow cytometry ( A ). Expression of Il21 ( B ) and Tbx21 ( C ) was determined by quantitative RT-PCR. Results were normalized using β-actin transcripts and represent relative expression (mean ± SEM, B , * P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Quantitative RT-PCR

Modulation of Th17 differentiation by CP-690,550. Sorted naïve CD4 + T cells were activated for 3 days with anti-CD3/anti-CD28 antibodies in the presence of IL-6 in combination with either TGF-β1, IL-23 or TGF-β1 and IL-23. CP-690,550 was added to the cultures at the indicated concentrations. Expression of IL-17A and IL-2 was determined by intracellular cytokine staining after stimulation with PMA/ionomycin. The effect of CP-690,550 on IL-17A expression was also studied in conditions, where TGF-β1 signaling was blocked by neutralizing antibodies. Representative flow cytometry plots ( A ) and summarized data of 3-4 separate experiments are shown ( B ). Expression of Rorc , Ahr and Il23r in cells activated as in A was determined by quantitative RT-PCR. Results were normalized using β-actin transcripts and represent fold increase of expression (mean ± SEM) compared to Th0 conditions ( C ).
Figure Legend Snippet: Modulation of Th17 differentiation by CP-690,550. Sorted naïve CD4 + T cells were activated for 3 days with anti-CD3/anti-CD28 antibodies in the presence of IL-6 in combination with either TGF-β1, IL-23 or TGF-β1 and IL-23. CP-690,550 was added to the cultures at the indicated concentrations. Expression of IL-17A and IL-2 was determined by intracellular cytokine staining after stimulation with PMA/ionomycin. The effect of CP-690,550 on IL-17A expression was also studied in conditions, where TGF-β1 signaling was blocked by neutralizing antibodies. Representative flow cytometry plots ( A ) and summarized data of 3-4 separate experiments are shown ( B ). Expression of Rorc , Ahr and Il23r in cells activated as in A was determined by quantitative RT-PCR. Results were normalized using β-actin transcripts and represent fold increase of expression (mean ± SEM) compared to Th0 conditions ( C ).

Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry, Quantitative RT-PCR

20) Product Images from "MALT1 Auto-Proteolysis Is Essential for NF-κB-Dependent Gene Transcription in Activated Lymphocytes"

Article Title: MALT1 Auto-Proteolysis Is Essential for NF-κB-Dependent Gene Transcription in Activated Lymphocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0103774

MALT1 auto-proteolysis is required for IL-2 production by Jurkat T cells. A) Jurkat T cells were left untreated or stimulated with P/I for 30 min, with or without pre min, with or without pre-treatment with 50 µM z-VRPR-fmk for 30 min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p-ERK (activation control) and tubulin (loading control). B) IL-2 production (ELISA) of Jurkat T cells stably expressing MALT1, MALT1-R149A, MALT1-C464A or MALT1-RACA, either untreated (-) or stimulated for 18 hrs with PMA hrs with PMA/ionomycin (P/I). Data shown as mean +/- S.D. (n = 3). Inset: Immunoblot with a-MALT1-C and a-Flag showing expression of ectopic MALT1 and mutants relative to endogenous MALT1 (lane 1). Numbers indicate fold overexpression relative to endogenous MALT1. AS: a-specific band obtained with a-Flag that serves as loading control. C) Immunoblot of cell lysates (top) and bio-IPs (bottom) from Jurkat T cells and Jurkat T cells with stable expression of Avi-tagged MALT1 or MALT1 mutants R149A, C464A and RACA with indicated antibodies.
Figure Legend Snippet: MALT1 auto-proteolysis is required for IL-2 production by Jurkat T cells. A) Jurkat T cells were left untreated or stimulated with P/I for 30 min, with or without pre min, with or without pre-treatment with 50 µM z-VRPR-fmk for 30 min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p-ERK (activation control) and tubulin (loading control). B) IL-2 production (ELISA) of Jurkat T cells stably expressing MALT1, MALT1-R149A, MALT1-C464A or MALT1-RACA, either untreated (-) or stimulated for 18 hrs with PMA hrs with PMA/ionomycin (P/I). Data shown as mean +/- S.D. (n = 3). Inset: Immunoblot with a-MALT1-C and a-Flag showing expression of ectopic MALT1 and mutants relative to endogenous MALT1 (lane 1). Numbers indicate fold overexpression relative to endogenous MALT1. AS: a-specific band obtained with a-Flag that serves as loading control. C) Immunoblot of cell lysates (top) and bio-IPs (bottom) from Jurkat T cells and Jurkat T cells with stable expression of Avi-tagged MALT1 or MALT1 mutants R149A, C464A and RACA with indicated antibodies.

Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, Over Expression

MALT1 auto-proteolysis is required for NF-κB transcriptional activity in Jurkat T cells. A) Relative IL-2 and CSF2 production of indicated Jurkat T cell lines, stimulated for 18 hrs with PMA hrs with PMA/ionomycin, measured via ELISA. Data shown as mean +/- S.D. (n = 3). B) Jurkat T cells expressing MALT1 and the JΔM-CA, JΔM-RA, and JΔM cells were stimulated for the indicated times with P/I and IL-2 and CSF2 transcript levels were determined via qRT-PCR. C) Jurkat T cells with ectopic expression of MALT1 and JΔM-CA, JΔM-RA and JΔM were electroporated (Amaxa, Nucleofection) with Luciferase reporter constructs driven by the IL-2 promoter or the Igκ3-ConA promoter, and 24 hrs later stimulated with P hrs later stimulated with P/I for 18 hrs before Luciferase activity was measured. Data shown as mean +/- S.D. (n = 3). D) GSEA showing a significant enrichment of NF-κB-target genes (FDR q
Figure Legend Snippet: MALT1 auto-proteolysis is required for NF-κB transcriptional activity in Jurkat T cells. A) Relative IL-2 and CSF2 production of indicated Jurkat T cell lines, stimulated for 18 hrs with PMA hrs with PMA/ionomycin, measured via ELISA. Data shown as mean +/- S.D. (n = 3). B) Jurkat T cells expressing MALT1 and the JΔM-CA, JΔM-RA, and JΔM cells were stimulated for the indicated times with P/I and IL-2 and CSF2 transcript levels were determined via qRT-PCR. C) Jurkat T cells with ectopic expression of MALT1 and JΔM-CA, JΔM-RA and JΔM were electroporated (Amaxa, Nucleofection) with Luciferase reporter constructs driven by the IL-2 promoter or the Igκ3-ConA promoter, and 24 hrs later stimulated with P hrs later stimulated with P/I for 18 hrs before Luciferase activity was measured. Data shown as mean +/- S.D. (n = 3). D) GSEA showing a significant enrichment of NF-κB-target genes (FDR q

Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Luciferase, Construct

Figure 5. MALT1 auto-proteolysis in activated B cells. A) ABC-DLBCL cell lines HBL-1 and OCI-Ly3 were treated with 50 µM z-VRPR-fmk (36 hrs hrs) and lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments with a-MALT1, a-Cleaved BCL10 and a-Tubulin (loading control). B-C) The GCB-DLBCL cell lines BJAB and Raji were left untreated or stimulated with PMA/ionomycin (30 min min) with or without pre-treatment with 50 µM z-VRPR-fmk (30 min min). Lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments, for p-ERK (activation control) and tubulin (loading control). D) Immunoblot of lysates of SSK41 cells and SSK41 cells with ectopic expression of the API2-MALT1 fusion variants A7M3 and A7M8, or the L232LI mutant of Card11 (C11m) respectively, with antibodies against the MALT1 C-terminus, the p76 neo-epitope, the CYLD C-terminus, the NIK C-terminus and Flag (ectopic A7M3, A7M8 and C11m). Numbers below blots depict band intensities of MALT1, p76 and the CYLD p70 fragment relative to lane 1. * = non-specific band. LC: loading control, a non-specific band obtained with the p76 neo-epitope antibody was used.
Figure Legend Snippet: Figure 5. MALT1 auto-proteolysis in activated B cells. A) ABC-DLBCL cell lines HBL-1 and OCI-Ly3 were treated with 50 µM z-VRPR-fmk (36 hrs hrs) and lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments with a-MALT1, a-Cleaved BCL10 and a-Tubulin (loading control). B-C) The GCB-DLBCL cell lines BJAB and Raji were left untreated or stimulated with PMA/ionomycin (30 min min) with or without pre-treatment with 50 µM z-VRPR-fmk (30 min min). Lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments, for p-ERK (activation control) and tubulin (loading control). D) Immunoblot of lysates of SSK41 cells and SSK41 cells with ectopic expression of the API2-MALT1 fusion variants A7M3 and A7M8, or the L232LI mutant of Card11 (C11m) respectively, with antibodies against the MALT1 C-terminus, the p76 neo-epitope, the CYLD C-terminus, the NIK C-terminus and Flag (ectopic A7M3, A7M8 and C11m). Numbers below blots depict band intensities of MALT1, p76 and the CYLD p70 fragment relative to lane 1. * = non-specific band. LC: loading control, a non-specific band obtained with the p76 neo-epitope antibody was used.

Techniques Used: Activation Assay, Expressing, Mutagenesis

21) Product Images from "Tumor Expression of CD200 Inhibits IL-10 Production by Tumor-Associated Myeloid Cells and Prevents Tumor Immune Evasion of CTL Therapy"

Article Title: Tumor Expression of CD200 Inhibits IL-10 Production by Tumor-Associated Myeloid Cells and Prevents Tumor Immune Evasion of CTL Therapy

Journal: European journal of immunology

doi: 10.1002/eji.201040472

Role of TAMC-derived IL-10 in suppression of CTL activity. Spleen and lymph node cells from RAG2 −/− P1CTL mice were co-cultured with CD11b + cells from J558-Ctrl tumors in the presence of P1A35-43. 5 µg/ml of anti-IL-10 antibody or an isotype matched control antibody were added in the culture (left panel). CD11b + cells from BALB/c or IL-10 −/− BALB/c mice with J558-Ctrl tumors were compared for their suppressive activity using thymidine incorporation assay (right panel). Suppressor: effector =1:1. b. 0.3 × 10 6 /ml CD11b + cells from WT or IL-10−/− mice with J558-Ctrl tumors were stimulated with 100 ng/ml of PMA and 100 ng/ml of ionomycin for 24 h. TNF-α production in the culture supernatants were detected by ELISA. c. Effect of P1CTL treatment of mice with established J558-Ctrl tumors. 5 × 10 6 of CD8 + T cells from P1CTL transgenic mice were injected into each mouse with established J558-ctrl tumors. Numbers of mice with recurrent tumors were significantly different between the two groups (P
Figure Legend Snippet: Role of TAMC-derived IL-10 in suppression of CTL activity. Spleen and lymph node cells from RAG2 −/− P1CTL mice were co-cultured with CD11b + cells from J558-Ctrl tumors in the presence of P1A35-43. 5 µg/ml of anti-IL-10 antibody or an isotype matched control antibody were added in the culture (left panel). CD11b + cells from BALB/c or IL-10 −/− BALB/c mice with J558-Ctrl tumors were compared for their suppressive activity using thymidine incorporation assay (right panel). Suppressor: effector =1:1. b. 0.3 × 10 6 /ml CD11b + cells from WT or IL-10−/− mice with J558-Ctrl tumors were stimulated with 100 ng/ml of PMA and 100 ng/ml of ionomycin for 24 h. TNF-α production in the culture supernatants were detected by ELISA. c. Effect of P1CTL treatment of mice with established J558-Ctrl tumors. 5 × 10 6 of CD8 + T cells from P1CTL transgenic mice were injected into each mouse with established J558-ctrl tumors. Numbers of mice with recurrent tumors were significantly different between the two groups (P

Techniques Used: Derivative Assay, CTL Assay, Activity Assay, Mouse Assay, Cell Culture, Thymidine Incorporation Assay, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Injection

Tumor infiltrating CTL preferentially accumulate in CD200-positive tumors and are capable of producing more IFN-γ. Purified P1CTL were injected into RAG-2 −/− BALB/c mice with established J558-CD200 or J558-Ctrl tumors. At various times after P1CTL injection, single cell suspensions were prepared from tumors and were stained for Vα8.3, CD8 and/or IFN-γ. For IFN-γ staining, cells were stimulated with 50 ng/ml of PMA/Ionomycin for 5 h followed by a standard intracellular staining procedure. a. Flow cytometry analysis of frequencies of tumor antigen-specific T cells in CD200-positive and CD200-negative tumors. b. Numbers of tumor-infiltrating P1CTL at different times after injection. Data show mean + SD (n= 3–7 mice). c. Flow cytometry analysis of IFN-γ production by tumor infiltrating P1CTL. d. Quantitation of tumor-infiltrating P1CTL that were IFN-γ positive. Data show mean + SD (n=3–7 mice). Data shown in panels c and d were at day 5 after P1CTL transfer. Paired student’s t test was used for the statistical analysis.
Figure Legend Snippet: Tumor infiltrating CTL preferentially accumulate in CD200-positive tumors and are capable of producing more IFN-γ. Purified P1CTL were injected into RAG-2 −/− BALB/c mice with established J558-CD200 or J558-Ctrl tumors. At various times after P1CTL injection, single cell suspensions were prepared from tumors and were stained for Vα8.3, CD8 and/or IFN-γ. For IFN-γ staining, cells were stimulated with 50 ng/ml of PMA/Ionomycin for 5 h followed by a standard intracellular staining procedure. a. Flow cytometry analysis of frequencies of tumor antigen-specific T cells in CD200-positive and CD200-negative tumors. b. Numbers of tumor-infiltrating P1CTL at different times after injection. Data show mean + SD (n= 3–7 mice). c. Flow cytometry analysis of IFN-γ production by tumor infiltrating P1CTL. d. Quantitation of tumor-infiltrating P1CTL that were IFN-γ positive. Data show mean + SD (n=3–7 mice). Data shown in panels c and d were at day 5 after P1CTL transfer. Paired student’s t test was used for the statistical analysis.

Techniques Used: CTL Assay, Purification, Injection, Mouse Assay, Staining, Flow Cytometry, Cytometry, Quantitation Assay

Role of IL-10 produced by TAMC. a . Analysis of TAMC-associated factors by real-time PCR. RNA samples were prepared from established J558-CD200 and J558-ctrl tumors (the hosts were RAG-2 −/− BALB/c mice). Expression of genes associated with TAMC (IL-10 and TNF-α) was quantitated. b. CD11b + cells from J558-CD200 or J558-Ctrl tumors were stimulated with100 ng/ml of PMA and ionomycin for 24 h, IL-10 and TNF-α releases in the culture supernatants were measured by ELISA. c. CD11b + cells in spleens and tumors from mice bearing J558-Ctrl tumors were co-cultured with either J558-Ctrl or J558-CD200 cells at a 1:1 ratio for 24 h in the presence of 100 ng/ml of PMA and ionomycin. IL-10 concentrations in the culture supernatants were then detected by ELISA. Data are normalized to 1 × 10 6 /ml of TAMC, show mean + SD (n=3) and are representative of three independent experiments. Student’s t test was used for the statistical analysis.
Figure Legend Snippet: Role of IL-10 produced by TAMC. a . Analysis of TAMC-associated factors by real-time PCR. RNA samples were prepared from established J558-CD200 and J558-ctrl tumors (the hosts were RAG-2 −/− BALB/c mice). Expression of genes associated with TAMC (IL-10 and TNF-α) was quantitated. b. CD11b + cells from J558-CD200 or J558-Ctrl tumors were stimulated with100 ng/ml of PMA and ionomycin for 24 h, IL-10 and TNF-α releases in the culture supernatants were measured by ELISA. c. CD11b + cells in spleens and tumors from mice bearing J558-Ctrl tumors were co-cultured with either J558-Ctrl or J558-CD200 cells at a 1:1 ratio for 24 h in the presence of 100 ng/ml of PMA and ionomycin. IL-10 concentrations in the culture supernatants were then detected by ELISA. Data are normalized to 1 × 10 6 /ml of TAMC, show mean + SD (n=3) and are representative of three independent experiments. Student’s t test was used for the statistical analysis.

Techniques Used: Produced, Real-time Polymerase Chain Reaction, Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

22) Product Images from "CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells"

Article Title: CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20021429

(a) H-Y–specific T cells recovered from CD28WT d18 pregnants exhibit decrease proliferation to H-Y peptide. 4 × 10 5 CFSE-labeled spleen cells from either nonpregnant or d18 pregnant H-Y CD28WT mice were cultured with 0.1 nM peptide for 72 h. T cells from pregnancies with mixed litters were compared with pregnancies with only females in the litter. (b) H-Y–specific T cells recovered from CD28KO pregnants do not exhibit decreased proliferation to H-Y peptide or male APCs. 4 × 10 5 CFSE-labeled spleen cells from either nonpregnant or d18 pregnant H-Y CD28WT or KO mice were cultured with 0.1 nM peptide for 72 h. Proliferation was assessed by dye dilution after gating on T3.70 + Annexin V − cells (nonpregnant, n = 28; d18, n = 6; d18 females only, n = 3; CD28KO nonpregnant, n = 16; CD28WT d18, n = 12). (c) Fewer H-Y–specific T cells recovered from CD28KO pregnants secrete IFN-γ. 4 × 10 5 spleen cells from CD28WT or CD28KO H-Y mice were cultured in the presence of 1,000 nM or 1 nM peptide for 72 h, then harvested and cultured for an additional 6 h in the presence of GolgiStop, 200 ng/ml PMA, and 750 ng/ml ionomycin. Clonotypic T cells were analyzed for intracellular expression of IFN-γ. Similar results were obtained in experiments restimulating with peptide instead of PMA and ionomycin. (d) H-Y–specific T cells from CD28KO d18 pregnants do not exhibit decreased CTL activity. 4 × 10 6 CFSE-labeled spleen cells from CD28WT or CD28KO H-Y mice were cultured with 1 μM peptide for 72 h, harvested, and cultured with EL-4 cells in the absence or presence of peptide for 4 h. EL-4 target cells were then analyzed for expression of Annexin V by flow cytometry. Results represent pooled data from multiple experiments (CD28WT nonpregnant, n = 9; CD28WT d18, n = 3; CD28KO nonpregnant, n = 6; CD28KO d18, n = 4).
Figure Legend Snippet: (a) H-Y–specific T cells recovered from CD28WT d18 pregnants exhibit decrease proliferation to H-Y peptide. 4 × 10 5 CFSE-labeled spleen cells from either nonpregnant or d18 pregnant H-Y CD28WT mice were cultured with 0.1 nM peptide for 72 h. T cells from pregnancies with mixed litters were compared with pregnancies with only females in the litter. (b) H-Y–specific T cells recovered from CD28KO pregnants do not exhibit decreased proliferation to H-Y peptide or male APCs. 4 × 10 5 CFSE-labeled spleen cells from either nonpregnant or d18 pregnant H-Y CD28WT or KO mice were cultured with 0.1 nM peptide for 72 h. Proliferation was assessed by dye dilution after gating on T3.70 + Annexin V − cells (nonpregnant, n = 28; d18, n = 6; d18 females only, n = 3; CD28KO nonpregnant, n = 16; CD28WT d18, n = 12). (c) Fewer H-Y–specific T cells recovered from CD28KO pregnants secrete IFN-γ. 4 × 10 5 spleen cells from CD28WT or CD28KO H-Y mice were cultured in the presence of 1,000 nM or 1 nM peptide for 72 h, then harvested and cultured for an additional 6 h in the presence of GolgiStop, 200 ng/ml PMA, and 750 ng/ml ionomycin. Clonotypic T cells were analyzed for intracellular expression of IFN-γ. Similar results were obtained in experiments restimulating with peptide instead of PMA and ionomycin. (d) H-Y–specific T cells from CD28KO d18 pregnants do not exhibit decreased CTL activity. 4 × 10 6 CFSE-labeled spleen cells from CD28WT or CD28KO H-Y mice were cultured with 1 μM peptide for 72 h, harvested, and cultured with EL-4 cells in the absence or presence of peptide for 4 h. EL-4 target cells were then analyzed for expression of Annexin V by flow cytometry. Results represent pooled data from multiple experiments (CD28WT nonpregnant, n = 9; CD28WT d18, n = 3; CD28KO nonpregnant, n = 6; CD28KO d18, n = 4).

Techniques Used: Labeling, Mouse Assay, Cell Culture, Expressing, CTL Assay, Activity Assay, Flow Cytometry, Cytometry

23) Product Images from "Vaccination with Embryonic Stem Cells Protects against Lung Cancer: Is a Broad-Spectrum Prophylactic Vaccine against Cancer Possible?"

Article Title: Vaccination with Embryonic Stem Cells Protects against Lung Cancer: Is a Broad-Spectrum Prophylactic Vaccine against Cancer Possible?

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042289

ESC vaccination induces tumor cell-specific, Th1-mediated cytokine response in CD8 + T cells. C57BL/6 mice (6/group) were immunized twice (days 0 and 14) with HBSS (control) or irradiated 1×10 6 ESC alone, or irradiated 1×10 6 ESC+irradiated 1×10 6 STO-GM, or irradiated 1×10 6 STO-GM cells alone, s.c. in the right flank. Ten days after the boost, mice were euthanized and spleens were removed. Splenocytes from vaccinated and control mice were co-cultured with LLC lysate (50 µg/ml) for an additional 4 days. Effectors were harvested and stimulated for 4 hours with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of Brefeldin A (1 µl/ml). After restimulation, effectors were harvested, Fc receptors were blocked, and stained for surface expression of CD4, CD8 and intracellular expression of cytokines and analyzed by flow cytometry. ( A, B ) Dot plots showing TNF-α and IFN-γ expression in CD8 + cells in splenocyte cultures obtained from control and ESC/STO-GM vaccinated mice. Numbers in quadrants represent the percentages of each subpopulation. ( C, D ) Bar graphs showing percentages of CD8 + TNF-α + , CD8 + IFN-γ + , and CD8 + IL2 + cells in splenocyte cultures derived from control, ESC alone, STO-GM alone and ESC/STO-GM vaccinated mice. Results are expressed as percentages of total cells. *, p
Figure Legend Snippet: ESC vaccination induces tumor cell-specific, Th1-mediated cytokine response in CD8 + T cells. C57BL/6 mice (6/group) were immunized twice (days 0 and 14) with HBSS (control) or irradiated 1×10 6 ESC alone, or irradiated 1×10 6 ESC+irradiated 1×10 6 STO-GM, or irradiated 1×10 6 STO-GM cells alone, s.c. in the right flank. Ten days after the boost, mice were euthanized and spleens were removed. Splenocytes from vaccinated and control mice were co-cultured with LLC lysate (50 µg/ml) for an additional 4 days. Effectors were harvested and stimulated for 4 hours with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of Brefeldin A (1 µl/ml). After restimulation, effectors were harvested, Fc receptors were blocked, and stained for surface expression of CD4, CD8 and intracellular expression of cytokines and analyzed by flow cytometry. ( A, B ) Dot plots showing TNF-α and IFN-γ expression in CD8 + cells in splenocyte cultures obtained from control and ESC/STO-GM vaccinated mice. Numbers in quadrants represent the percentages of each subpopulation. ( C, D ) Bar graphs showing percentages of CD8 + TNF-α + , CD8 + IFN-γ + , and CD8 + IL2 + cells in splenocyte cultures derived from control, ESC alone, STO-GM alone and ESC/STO-GM vaccinated mice. Results are expressed as percentages of total cells. *, p

Techniques Used: Mouse Assay, Irradiation, Cell Culture, Staining, Expressing, Flow Cytometry, Cytometry, Derivative Assay

24) Product Images from "Identification of a gain-of-function STAT3 mutation (p.Y640F) in lymphocytic variant hypereosinophilic syndrome"

Article Title: Identification of a gain-of-function STAT3 mutation (p.Y640F) in lymphocytic variant hypereosinophilic syndrome

Journal: Blood

doi: 10.1182/blood-2015-06-654277

Activation of STAT3 gene targets in L-HES clones. (A) The mean fold-change of transcript levels (log2) in L-HES clones compared with CD4 + T-cell controls (control memory [CM]) as evaluated through RNA sequencing. L-HES clone 1 possesses the p.Y640F mutation. *** P = .0002; ** P = .0007; * P = .008. (B) Gene set enrichment analysis of STAT3 gene signature in L-HES clones compared with CD4 + T-cell controls ( P = .002). (C) Relative levels of messenger RNA (mRNA) transcripts of known STAT3 gene targets in unstimulated L-HES clones as compared with CD4 + T-cell controls. (D) Relative levels of cytokine expression in L-HES clones as compared with CD4 + T-cell controls. For panels C-D, closed symbols represent the L-HES clone containing the p.Y640F mutation. In panel D cytokine profiles, unstimulated L-HES clones were not annotated with closed symbols for the sake of clarity. Each T-cell sample was cultured with or without pharmacologic stimulation with phorbol 12-myristate 13-acetate and ionomycin. * P
Figure Legend Snippet: Activation of STAT3 gene targets in L-HES clones. (A) The mean fold-change of transcript levels (log2) in L-HES clones compared with CD4 + T-cell controls (control memory [CM]) as evaluated through RNA sequencing. L-HES clone 1 possesses the p.Y640F mutation. *** P = .0002; ** P = .0007; * P = .008. (B) Gene set enrichment analysis of STAT3 gene signature in L-HES clones compared with CD4 + T-cell controls ( P = .002). (C) Relative levels of messenger RNA (mRNA) transcripts of known STAT3 gene targets in unstimulated L-HES clones as compared with CD4 + T-cell controls. (D) Relative levels of cytokine expression in L-HES clones as compared with CD4 + T-cell controls. For panels C-D, closed symbols represent the L-HES clone containing the p.Y640F mutation. In panel D cytokine profiles, unstimulated L-HES clones were not annotated with closed symbols for the sake of clarity. Each T-cell sample was cultured with or without pharmacologic stimulation with phorbol 12-myristate 13-acetate and ionomycin. * P

Techniques Used: Activation Assay, Clone Assay, RNA Sequencing Assay, Mutagenesis, Expressing, Cell Culture

Identification of a p.Y640F STAT3 mutation in a patient with L-HES. (A) Clinical photographs of eosinophil-infiltrated papules and nodules on the patient’s trunk (upper) and elbow (lower). (B) Table of relevant laboratory findings, with reference values. (C) Intracellular cytokine staining of CD3 − CD4 + T cells. Peripheral blood mononuclear cells were stimulated for 5 hours with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (1 μg/mL). Cells were subsequently fixed, permeabilized, and stained with a phycoerythrin/cyanin 7–labeled anti-human CD3 antibody (BD Pharmingen) and an antibody cocktail containing antibodies for CD4, IL-4, IL-17A, and interferon-γ (IFNγ) (BD Pharmingen). (D) Confirmation of the somatic STAT3 mutation in CD3 − CD4 + T cells by Sanger sequencing. Matched normal CD3 + CD19 − B cells were used as a control.
Figure Legend Snippet: Identification of a p.Y640F STAT3 mutation in a patient with L-HES. (A) Clinical photographs of eosinophil-infiltrated papules and nodules on the patient’s trunk (upper) and elbow (lower). (B) Table of relevant laboratory findings, with reference values. (C) Intracellular cytokine staining of CD3 − CD4 + T cells. Peripheral blood mononuclear cells were stimulated for 5 hours with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (1 μg/mL). Cells were subsequently fixed, permeabilized, and stained with a phycoerythrin/cyanin 7–labeled anti-human CD3 antibody (BD Pharmingen) and an antibody cocktail containing antibodies for CD4, IL-4, IL-17A, and interferon-γ (IFNγ) (BD Pharmingen). (D) Confirmation of the somatic STAT3 mutation in CD3 − CD4 + T cells by Sanger sequencing. Matched normal CD3 + CD19 − B cells were used as a control.

Techniques Used: Mutagenesis, Staining, Labeling, Sequencing

25) Product Images from "The scaffolding function of the RLTPR protein explains its essential role for CD28 co-stimulation in mouse and human T cells"

Article Title: The scaffolding function of the RLTPR protein explains its essential role for CD28 co-stimulation in mouse and human T cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20160579

The mouse RLTPR CPI motif is dispensable for co-stimulation via CD28 and the development of T reg cells. (A) Cellularity of thymus and spleen of WT, Rltpr dcpi/dcpi , and Rltpr −/− mice (key: top right corner). (B) Quantification of T cells in the spleen of WT, Rltpr dcpi/dcpi , and Rltpr −/− mice. (C) Percent T reg cells in WT, Rltpr dcpi/dcpi , and Rltpr −/− spleen. (D) Percent CD4 + effector memory (CD44 + CD62L lo ) cells in WT, Rltpr dcpi/dcpi , and Rltpr −/− spleen. (E) ATP content of negatively purified CD4 + T cells from lymph nodes of WT, Rltpr dcpi/dcpi , and Rltpr −/− mice, activated for 48 h in vitro with plate-bound anti-CD3 in the presence or absence of soluble anti-CD28 or with PMA and ionomycin (PI), assessed as luminescence. (F) IL-2 in supernatants of purified WT, Rltpr dcpi/dcpi , and Rltpr −/− CD4 + T cells activated for 48 h, as in E. (G) Internalization of CD28 by WT, Rltpr dcpi/dcpi , Rltpr bas/bas , and Rltpr −/− peripheral T cells after cross-linkage for 5, 10, or 15 min. Results are presented as percent change in surface CD28 expression relative to that at time 0. Data representative of two experiments with three mice per group (mean and SEM). **, P ≤ 0.01; ***, P ≤ 0.005; ****, P ≤ 0.001; ns, nonsignificant.
Figure Legend Snippet: The mouse RLTPR CPI motif is dispensable for co-stimulation via CD28 and the development of T reg cells. (A) Cellularity of thymus and spleen of WT, Rltpr dcpi/dcpi , and Rltpr −/− mice (key: top right corner). (B) Quantification of T cells in the spleen of WT, Rltpr dcpi/dcpi , and Rltpr −/− mice. (C) Percent T reg cells in WT, Rltpr dcpi/dcpi , and Rltpr −/− spleen. (D) Percent CD4 + effector memory (CD44 + CD62L lo ) cells in WT, Rltpr dcpi/dcpi , and Rltpr −/− spleen. (E) ATP content of negatively purified CD4 + T cells from lymph nodes of WT, Rltpr dcpi/dcpi , and Rltpr −/− mice, activated for 48 h in vitro with plate-bound anti-CD3 in the presence or absence of soluble anti-CD28 or with PMA and ionomycin (PI), assessed as luminescence. (F) IL-2 in supernatants of purified WT, Rltpr dcpi/dcpi , and Rltpr −/− CD4 + T cells activated for 48 h, as in E. (G) Internalization of CD28 by WT, Rltpr dcpi/dcpi , Rltpr bas/bas , and Rltpr −/− peripheral T cells after cross-linkage for 5, 10, or 15 min. Results are presented as percent change in surface CD28 expression relative to that at time 0. Data representative of two experiments with three mice per group (mean and SEM). **, P ≤ 0.01; ***, P ≤ 0.005; ****, P ≤ 0.001; ns, nonsignificant.

Techniques Used: Mouse Assay, Purification, In Vitro, Expressing

The Rltpr bas mutation phenocopies a Rltpr -null mutation. (A) Cellularity of thymus and spleen of WT, Rltpr −/− , and Rltpr bas/bas mice (key: lower right corner). (B) Quantification of T cells in the spleen of WT, Rltpr −/− , and Rltpr bas/bas mice. (C) Expression of RLTPR (left) and CD28 (right) in double-positive thymocytes (DP) and splenic CD4 + T cells (CD4) from WT, Rltpr −/− , and Rltpr bas/bas mice, analyzed by flow cytometry (key: lower right corner). (D). CD4 + T cells from WT, Rltpr −/− , and Rltpr bas/bas spleens were analyzed for expression of Foxp3 and CD25. Numbers in quadrants indicate percent T reg cells, and quantification of T reg cells is shown on the right. (E) CD4 + T cells from WT, Rltpr −/− , and Rltpr bas/bas spleens were analyzed for expression of CD44 and CD62L. Numbers in quadrants indicate percent naive (CD44 lo CD62L hi ) and effector memory (CD44 hi CD62L lo ) CD4 + T cells. Histogram on the right shows percent effector memory CD4 + T cells. (F) ATP content of negatively purified CD4 + T cells from lymph nodes of WT, Rltpr −/− , and Rltpr bas/bas mice, activated for 48 h in vitro with plate-bound anti-CD3 in the presence or absence of soluble anti-CD28 or with PMA and ionomycin (PI). ATP content was assessed using luminescence as a measure of the extent of cell proliferation. (G) IL-2 in supernatants of purified WT, Rltpr −/− , and Rltpr bas/bas CD4 + T cells activated for 48 h as in F. Data are representative of at least three independent experiments, with two to three mice per genotype (D–G; mean and SEM). **, P ≤ 0.01; ***, P ≤ 0.005; ns, nonsignificant.
Figure Legend Snippet: The Rltpr bas mutation phenocopies a Rltpr -null mutation. (A) Cellularity of thymus and spleen of WT, Rltpr −/− , and Rltpr bas/bas mice (key: lower right corner). (B) Quantification of T cells in the spleen of WT, Rltpr −/− , and Rltpr bas/bas mice. (C) Expression of RLTPR (left) and CD28 (right) in double-positive thymocytes (DP) and splenic CD4 + T cells (CD4) from WT, Rltpr −/− , and Rltpr bas/bas mice, analyzed by flow cytometry (key: lower right corner). (D). CD4 + T cells from WT, Rltpr −/− , and Rltpr bas/bas spleens were analyzed for expression of Foxp3 and CD25. Numbers in quadrants indicate percent T reg cells, and quantification of T reg cells is shown on the right. (E) CD4 + T cells from WT, Rltpr −/− , and Rltpr bas/bas spleens were analyzed for expression of CD44 and CD62L. Numbers in quadrants indicate percent naive (CD44 lo CD62L hi ) and effector memory (CD44 hi CD62L lo ) CD4 + T cells. Histogram on the right shows percent effector memory CD4 + T cells. (F) ATP content of negatively purified CD4 + T cells from lymph nodes of WT, Rltpr −/− , and Rltpr bas/bas mice, activated for 48 h in vitro with plate-bound anti-CD3 in the presence or absence of soluble anti-CD28 or with PMA and ionomycin (PI). ATP content was assessed using luminescence as a measure of the extent of cell proliferation. (G) IL-2 in supernatants of purified WT, Rltpr −/− , and Rltpr bas/bas CD4 + T cells activated for 48 h as in F. Data are representative of at least three independent experiments, with two to three mice per genotype (D–G; mean and SEM). **, P ≤ 0.01; ***, P ≤ 0.005; ns, nonsignificant.

Techniques Used: Mutagenesis, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Purification, In Vitro

The lack of CD28 co-stimulation in Rltpr bas/bas mice results from the L432P mutation itself and not from the coincidently diminished levels of RLTPR bas protein. (A) Expression of RLTPR (left) and CD28 (right) in splenic CD4 + T cells from WT, Rltpr +/− , Rltpr −/− , and Rltpr bas/bas mice, analyzed by flow cytometry. (B) ATP content of negatively purified CD4 + T cells from lymph nodes of WT, Rltpr +/− , Rltpr −/− and Rltpr bas/bas mice, activated for 48 h in vitro with plate-bound anti-CD3 in the presence or absence of soluble anti-CD28 or with PMA and ionomycin (PI). ATP content was assessed using luminescence. (C) IL-2 in supernatants of purified WT, Rltpr +/− , Rltpr −/− , and Rltpr bas/bas CD4 + T cells activated for 48 h as in (B). Data are representative of at least two experiments with three mice per group (B and C; mean and SEM). ***, P ≤ 0.005; ns, nonsignificant. Gray shaded curves (A), isotype-matched control antibody (negative control).
Figure Legend Snippet: The lack of CD28 co-stimulation in Rltpr bas/bas mice results from the L432P mutation itself and not from the coincidently diminished levels of RLTPR bas protein. (A) Expression of RLTPR (left) and CD28 (right) in splenic CD4 + T cells from WT, Rltpr +/− , Rltpr −/− , and Rltpr bas/bas mice, analyzed by flow cytometry. (B) ATP content of negatively purified CD4 + T cells from lymph nodes of WT, Rltpr +/− , Rltpr −/− and Rltpr bas/bas mice, activated for 48 h in vitro with plate-bound anti-CD3 in the presence or absence of soluble anti-CD28 or with PMA and ionomycin (PI). ATP content was assessed using luminescence. (C) IL-2 in supernatants of purified WT, Rltpr +/− , Rltpr −/− , and Rltpr bas/bas CD4 + T cells activated for 48 h as in (B). Data are representative of at least two experiments with three mice per group (B and C; mean and SEM). ***, P ≤ 0.005; ns, nonsignificant. Gray shaded curves (A), isotype-matched control antibody (negative control).

Techniques Used: Mouse Assay, Mutagenesis, Expressing, Flow Cytometry, Cytometry, Purification, In Vitro, Negative Control

26) Product Images from "Silenced suppressor of cytokine signaling 1 (SOCS1) enhances the maturation and antifungal immunity of dendritic cells in response to Candida albicans in vitro"

Article Title: Silenced suppressor of cytokine signaling 1 (SOCS1) enhances the maturation and antifungal immunity of dendritic cells in response to Candida albicans in vitro

Journal: Immunologic Research

doi: 10.1007/s12026-014-8562-8

Exogenous anti-IL-12 or anti-IFN-γ mAbs reversed the enhancement of IFN-γ production by CD4 + T cells. a , b DCs were exposed to SOCS1 siRNA and were treated with C. albicans for 24 h. At day 10, T cells were stimulated with PMA and ionomycin for 5 h in the presence of brefeldin A. Cells were stained with anti-IFN-γ-FITC and IL-17-FITC to detect intracellular cytokine production by flow cytometry. Data were Mean ± SD of seven independent experiments. * p
Figure Legend Snippet: Exogenous anti-IL-12 or anti-IFN-γ mAbs reversed the enhancement of IFN-γ production by CD4 + T cells. a , b DCs were exposed to SOCS1 siRNA and were treated with C. albicans for 24 h. At day 10, T cells were stimulated with PMA and ionomycin for 5 h in the presence of brefeldin A. Cells were stained with anti-IFN-γ-FITC and IL-17-FITC to detect intracellular cytokine production by flow cytometry. Data were Mean ± SD of seven independent experiments. * p

Techniques Used: Staining, Flow Cytometry, Cytometry

27) Product Images from "SYK inhibitor entospletinib prevents ocular and skin GVHD in mice"

Article Title: SYK inhibitor entospletinib prevents ocular and skin GVHD in mice

Journal: JCI Insight

doi: 10.1172/jci.insight.122430

ENTO reduces activated B cells and Th2 cells in +Spl GVHD mice. ( A ) Representative flow cytometry plots showing gating strategies for GL7 expression by activated B cells and IL-4 expression by Th2 cells in splenocytes harvested from experimental mice and kept viably frozen until the time of analysis. To identify activated B cells, freshly thawed splenocytes were stained with B220 and GL7. To identify Th2 cells, splenocytes were cultured for 5 hours in medium in the presence of PMA and ionomycin and then surface stained for CD4, followed by intracellular staining for IL-4 (utilizing a Mouse BD Biosciences Th1/Th2/Th17 Phenotyping Kit). ( B ) Splenocytes from mice in all treatment groups ( n = 5 each) analyzed for the subsets described in A . Bars indicate the median ± range. Statistical analysis was performed by 1-way ANOVA with Tukey’s multiple comparisons test (GraphPad Prism). * P
Figure Legend Snippet: ENTO reduces activated B cells and Th2 cells in +Spl GVHD mice. ( A ) Representative flow cytometry plots showing gating strategies for GL7 expression by activated B cells and IL-4 expression by Th2 cells in splenocytes harvested from experimental mice and kept viably frozen until the time of analysis. To identify activated B cells, freshly thawed splenocytes were stained with B220 and GL7. To identify Th2 cells, splenocytes were cultured for 5 hours in medium in the presence of PMA and ionomycin and then surface stained for CD4, followed by intracellular staining for IL-4 (utilizing a Mouse BD Biosciences Th1/Th2/Th17 Phenotyping Kit). ( B ) Splenocytes from mice in all treatment groups ( n = 5 each) analyzed for the subsets described in A . Bars indicate the median ± range. Statistical analysis was performed by 1-way ANOVA with Tukey’s multiple comparisons test (GraphPad Prism). * P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Staining, Cell Culture

ENTO enhanced Tregs in +Spl GVHD mice. ( A ) Representative flow cytometry plots showing gating strategies for Foxp3 expression, cytokine production, and CXCR5 expression for the Treg, Th1, Th17, and Tfh subsets. To detect Tregs, splenocytes were surface stained for CD4, followed by intracellular staining for Foxp3. To detect Th1 and Th17 cells, splenocytes were cultured in the presence of PMA and ionomycin for 5 hours and then stained for surface CD4 and stained intracellularly for IFN-γ or IL-17 (utilizing a Mouse BD Biosciences Th1/Th2/Th17 Phenotyping Kit). To detect Tfh cells, splenocytes were surface stained for CD4 and CXCR5. ( B ) Splenocytes from mice in all treatment groups ( n = 5 each) analyzed for the subsets described in A . Bars indicate the median ± range. Statistical analysis was performed by 1-way ANOVA with Tukey’s multiple comparisons test (GraphPad Prism). * P
Figure Legend Snippet: ENTO enhanced Tregs in +Spl GVHD mice. ( A ) Representative flow cytometry plots showing gating strategies for Foxp3 expression, cytokine production, and CXCR5 expression for the Treg, Th1, Th17, and Tfh subsets. To detect Tregs, splenocytes were surface stained for CD4, followed by intracellular staining for Foxp3. To detect Th1 and Th17 cells, splenocytes were cultured in the presence of PMA and ionomycin for 5 hours and then stained for surface CD4 and stained intracellularly for IFN-γ or IL-17 (utilizing a Mouse BD Biosciences Th1/Th2/Th17 Phenotyping Kit). To detect Tfh cells, splenocytes were surface stained for CD4 and CXCR5. ( B ) Splenocytes from mice in all treatment groups ( n = 5 each) analyzed for the subsets described in A . Bars indicate the median ± range. Statistical analysis was performed by 1-way ANOVA with Tukey’s multiple comparisons test (GraphPad Prism). * P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Staining, Cell Culture

28) Product Images from "Restoration of regulatory and effector T cell balance and B cell homeostasis in systemic lupus erythematosus patients through vitamin D supplementation"

Article Title: Restoration of regulatory and effector T cell balance and B cell homeostasis in systemic lupus erythematosus patients through vitamin D supplementation

Journal: Arthritis Research & Therapy

doi: 10.1186/ar4060

Vitamin D supplementation induces a significant decrease in Th1 and Th17 cells . Peripheral blood mononuclear cells ( PBMCs) were stimulated for 4 hours with Phorbol myristate acetate (PMA) and ionomycin. After gating on CD3 + T cells, frequencies of IFN-γ-producing CD4 + (Th1) and CD8 + T cells, IL-17A-producing CD4 + T cells (Th17) and IL-4-producing CD4 + T cells (Th2) were analyzed with vitamin D supplementation. Th1, IFN-γ-producing CD8 + T cells and Th17 cells decreased after 2 months of vitamin D supplementation. Mean ± standard error of the mean (SEM) is shown. * P
Figure Legend Snippet: Vitamin D supplementation induces a significant decrease in Th1 and Th17 cells . Peripheral blood mononuclear cells ( PBMCs) were stimulated for 4 hours with Phorbol myristate acetate (PMA) and ionomycin. After gating on CD3 + T cells, frequencies of IFN-γ-producing CD4 + (Th1) and CD8 + T cells, IL-17A-producing CD4 + T cells (Th17) and IL-4-producing CD4 + T cells (Th2) were analyzed with vitamin D supplementation. Th1, IFN-γ-producing CD8 + T cells and Th17 cells decreased after 2 months of vitamin D supplementation. Mean ± standard error of the mean (SEM) is shown. * P

Techniques Used:

29) Product Images from "IL-17A inhibitions of indole alkaloids from traditional Chinese medicine Qing Dai"

Article Title: IL-17A inhibitions of indole alkaloids from traditional Chinese medicine Qing Dai

Journal: Journal of Ethnopharmacology

doi: 10.1016/j.jep.2020.112772

Th17 cells cytotoxicity (A, C) and IL-17 protein expression (B, D) data of compound 2. For the co-treated experiment, CD4 + T cells were cultured for five days with or without compound 2 in the skewing medium during Th17 cell polarization. After five days, cell viability was measured by staining with 1 μg/mL propidium iodide (A). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (B). For the post-treated experiment, the polarized Th17 cells were treated with or without compound 2 for 16 h. After incubation, cells were restimulated with PMA and ionomycin for 5 h, and then cell viability was measured by staining with 1 μg/mL propidium iodide (C). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (D). The data shown are representative of three independent experiments. All statistical tests were performed by the student's t-test at the two-tailed significance level of 0.05. * p
Figure Legend Snippet: Th17 cells cytotoxicity (A, C) and IL-17 protein expression (B, D) data of compound 2. For the co-treated experiment, CD4 + T cells were cultured for five days with or without compound 2 in the skewing medium during Th17 cell polarization. After five days, cell viability was measured by staining with 1 μg/mL propidium iodide (A). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (B). For the post-treated experiment, the polarized Th17 cells were treated with or without compound 2 for 16 h. After incubation, cells were restimulated with PMA and ionomycin for 5 h, and then cell viability was measured by staining with 1 μg/mL propidium iodide (C). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (D). The data shown are representative of three independent experiments. All statistical tests were performed by the student's t-test at the two-tailed significance level of 0.05. * p

Techniques Used: Expressing, Cell Culture, Staining, Flow Cytometry, Incubation, Two Tailed Test

Compounds 1 and 2 inhibited the IL-17 gene expression in the IL-17 luciferase reporter assay. IL-17Luc cells were stimulated with PMA and ionomycin, and co-treated with DMSO or indicating compounds for 5 h. Then the activity of luciferase in the transfected cells was measured with the microplate reader. Vector was control vector (pGL4.18) transfected cells. IL 17A was IL-17 promoter-luciferase gene (pGL4-hIL-17prom) transfected cells. P + I: PMA and ionomycin. The data shown are representative of three independent experiments. All statistical tests were performed by the student's t-test at the two-tailed significance level of 0.05. * p
Figure Legend Snippet: Compounds 1 and 2 inhibited the IL-17 gene expression in the IL-17 luciferase reporter assay. IL-17Luc cells were stimulated with PMA and ionomycin, and co-treated with DMSO or indicating compounds for 5 h. Then the activity of luciferase in the transfected cells was measured with the microplate reader. Vector was control vector (pGL4.18) transfected cells. IL 17A was IL-17 promoter-luciferase gene (pGL4-hIL-17prom) transfected cells. P + I: PMA and ionomycin. The data shown are representative of three independent experiments. All statistical tests were performed by the student's t-test at the two-tailed significance level of 0.05. * p

Techniques Used: Expressing, Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Two Tailed Test

Th17 cells cytotoxicity (A, C) and IL-17 protein expression (B, D) data of compound 1 . For the co-treated experiment, CD4 + T cells were cultured for five days with or without compound 1 in the skewing medium during Th17 cell polarization. After five days, cell viability was measured by staining with 1 μg/mL propidium iodide (A). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (B). For the post-treated experiment, the polarized Th17 cells were treated with or without compound 1 for 16 h. After incubation, cells were restimulated with PMA and ionomycin for 5 h, and then cell viability was measured by staining with 1 μg/mL propidium iodide (C). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (D). The data shown are representative of three independent experiments. All statistical tests were performed by the student's t-test at the two-tailed significance level of 0.05. ** p
Figure Legend Snippet: Th17 cells cytotoxicity (A, C) and IL-17 protein expression (B, D) data of compound 1 . For the co-treated experiment, CD4 + T cells were cultured for five days with or without compound 1 in the skewing medium during Th17 cell polarization. After five days, cell viability was measured by staining with 1 μg/mL propidium iodide (A). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (B). For the post-treated experiment, the polarized Th17 cells were treated with or without compound 1 for 16 h. After incubation, cells were restimulated with PMA and ionomycin for 5 h, and then cell viability was measured by staining with 1 μg/mL propidium iodide (C). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (D). The data shown are representative of three independent experiments. All statistical tests were performed by the student's t-test at the two-tailed significance level of 0.05. ** p

Techniques Used: Expressing, Cell Culture, Staining, Flow Cytometry, Incubation, Two Tailed Test

30) Product Images from "A predominant Th1 polarization is present in synovial fluid of end‐stage osteoarthritic knee joints: analysis of peripheral blood, synovial fluid and synovial membrane"

Article Title: A predominant Th1 polarization is present in synovial fluid of end‐stage osteoarthritic knee joints: analysis of peripheral blood, synovial fluid and synovial membrane

Journal: Clinical and Experimental Immunology

doi: 10.1111/cei.13230

Cytometric bead array (CBA) analysis of secreted cytokines in phorbol–myristate–acetate (PMA)/ionomycin‐stimulated T cell culture supernatants. Amounts of T cell secreted cytokines were analysed using the human T helper type 1 (Th1)/Th2/Th17 CBA kit (BD Biosciences) in PMA/ionomycin‐stimulated CD3 + CD8 – T cell culture supernatants of peripheral blood (PB), synovial fluid (SF) and synovial membrane (SM) from patients with OA ( n = 17). The work steps were performed according to the manufacturer’s instructions. Values in pg/ml are standardized for 1000 cells and shown in scatter dot‐plots as mean ± standard error of the mean (s.e.m.). Significant differences are marked with asterisks: * P
Figure Legend Snippet: Cytometric bead array (CBA) analysis of secreted cytokines in phorbol–myristate–acetate (PMA)/ionomycin‐stimulated T cell culture supernatants. Amounts of T cell secreted cytokines were analysed using the human T helper type 1 (Th1)/Th2/Th17 CBA kit (BD Biosciences) in PMA/ionomycin‐stimulated CD3 + CD8 – T cell culture supernatants of peripheral blood (PB), synovial fluid (SF) and synovial membrane (SM) from patients with OA ( n = 17). The work steps were performed according to the manufacturer’s instructions. Values in pg/ml are standardized for 1000 cells and shown in scatter dot‐plots as mean ± standard error of the mean (s.e.m.). Significant differences are marked with asterisks: * P

Techniques Used: Crocin Bleaching Assay, Cell Culture

31) Product Images from "Deoxynivalenol Has the Capacity to Increase Transcription Factor Expression and Cytokine Production in Porcine T Cells"

Article Title: Deoxynivalenol Has the Capacity to Increase Transcription Factor Expression and Cytokine Production in Porcine T Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.02009

Frequencies of IFN-γ and TNF-α producing cells in proliferating and non-proliferating CD4 + T cells in the presence of DON and DOM-1. Violet proliferation dye-stained PBMCs were cultivated for 4 days in the presence of ConA alone or in combination with two different DON concentrations (0.2 and 0.8 μM) and DOM-1 (16 μM). Following PMA/Ionomycin stimulation for 4 h on the fourth day, the cells were harvested and analyzed for cytokine production. (A) Representative flow cytometry data showing the gating of CD4 + T cells and analysis of total IFN-γ and TNF-α producing cells within proliferating (cells gated on the left) and non-proliferating (cells gated on the right) CD4 + T cells. Red numbers give percentages of cytokine producing cells. (B) Representative flow cytometry data showing the gating of proliferating and non-proliferating CD4 + T cells with subsequent analysis of IFN-γ single, TNF-α single, and IFN-γ/TNF-α co-producing CD4 + T cells in the presence of DON and DOM-1. Red numbers indicate percentages of cytokine-producing CD4 + T-cell subsets. (C) Boxplots summarize the data of experiments with PBMCs from six individual pigs and present the frequencies of proliferating and non-proliferating (parental) CD4 + T cells for the different cytokine producing subsets in the presence of DON and DOM-1. Different letters on boxplots indicate significant differences ( p
Figure Legend Snippet: Frequencies of IFN-γ and TNF-α producing cells in proliferating and non-proliferating CD4 + T cells in the presence of DON and DOM-1. Violet proliferation dye-stained PBMCs were cultivated for 4 days in the presence of ConA alone or in combination with two different DON concentrations (0.2 and 0.8 μM) and DOM-1 (16 μM). Following PMA/Ionomycin stimulation for 4 h on the fourth day, the cells were harvested and analyzed for cytokine production. (A) Representative flow cytometry data showing the gating of CD4 + T cells and analysis of total IFN-γ and TNF-α producing cells within proliferating (cells gated on the left) and non-proliferating (cells gated on the right) CD4 + T cells. Red numbers give percentages of cytokine producing cells. (B) Representative flow cytometry data showing the gating of proliferating and non-proliferating CD4 + T cells with subsequent analysis of IFN-γ single, TNF-α single, and IFN-γ/TNF-α co-producing CD4 + T cells in the presence of DON and DOM-1. Red numbers indicate percentages of cytokine-producing CD4 + T-cell subsets. (C) Boxplots summarize the data of experiments with PBMCs from six individual pigs and present the frequencies of proliferating and non-proliferating (parental) CD4 + T cells for the different cytokine producing subsets in the presence of DON and DOM-1. Different letters on boxplots indicate significant differences ( p

Techniques Used: Staining, Flow Cytometry

Frequencies of IFN-γ, TNF-α, and IL-17A producing CD2 + and CD2 – γδ T cells in the presence of DON and DOM-1. Violet proliferation dye-stained PBMCs were cultivated for 4 days in the presence of ConA alone or in combination with two different DON concentrations (0.2 and 0.8 μM) and DOM-1 (16 μM). Following PMA/Ionomycin stimulation for 4 h on the fourth day, the cells were harvested and analyzed for cytokine production. (A) CD2 + and CD2 – γδ T cells were gated and further analyzed for production of TNF-α, IFN-γ, and IL-17A in the presence of DON and DOM-1. The black rectangles in the pseudocolor plots show the gating strategy that was applied for analysis of total IFN-γ, total TNF-α, and total IL-17A producing cells. Red numbers give percentages of cytokine producing γδ T cells in these gates. Representative flow cytometry data from one animal is shown. (B,C) Boxplots show the results for γδ T cells within PBMCs from six individual pigs. (B) Frequency of IFN-γ and TNF-α producing cells within CD2 + γδ T cells. (C) Frequency of IFN-γ, TNF-α, and IL-17A producing cells within CD2 – γδ T cells. Different letters on boxplots indicate significant differences ( p
Figure Legend Snippet: Frequencies of IFN-γ, TNF-α, and IL-17A producing CD2 + and CD2 – γδ T cells in the presence of DON and DOM-1. Violet proliferation dye-stained PBMCs were cultivated for 4 days in the presence of ConA alone or in combination with two different DON concentrations (0.2 and 0.8 μM) and DOM-1 (16 μM). Following PMA/Ionomycin stimulation for 4 h on the fourth day, the cells were harvested and analyzed for cytokine production. (A) CD2 + and CD2 – γδ T cells were gated and further analyzed for production of TNF-α, IFN-γ, and IL-17A in the presence of DON and DOM-1. The black rectangles in the pseudocolor plots show the gating strategy that was applied for analysis of total IFN-γ, total TNF-α, and total IL-17A producing cells. Red numbers give percentages of cytokine producing γδ T cells in these gates. Representative flow cytometry data from one animal is shown. (B,C) Boxplots show the results for γδ T cells within PBMCs from six individual pigs. (B) Frequency of IFN-γ and TNF-α producing cells within CD2 + γδ T cells. (C) Frequency of IFN-γ, TNF-α, and IL-17A producing cells within CD2 – γδ T cells. Different letters on boxplots indicate significant differences ( p

Techniques Used: Staining, Flow Cytometry

Frequencies of IFN-γ, TNF-α, and IL-17A producing CD4 + T cells in the presence of DON and DOM-1. Violet proliferation dye-stained PBMCs were cultivated for 4 days in the presence of ConA alone or in combination with two different DON concentrations (0.2 and 0.8 μM) and DOM-1 (16 μM). Following PMA/Ionomycin stimulation for 4 h on the fourth day, the cells were harvested and analyzed for cytokine production. (A) Total CD4 + T cells were gated and analyzed for production of IFN-γ, TNF-α, and IL-17A. IFN-γ single producing T cells are designated with (1), single TNF-α with (2), total IFN-γ with (1 + 3), total TNF-α with (2 + 3), double IFN-γ + TNF-α + with (3), single IL-17A with (4), total IL-17A with (4 + 5), and double TNF-α + IL-17A + with (5). Representative flow cytometry data from one animal is shown. Red numbers indicate percentages of cytokine-producing CD4 + T-cell subsets. (B) Boxplots summarize the data of experiments with PBMCs from six pigs and show the frequencies of total IFN-γ, total TNF-α, and total IL-17A producing CD4 + T cells for the different DON and DOM-1 concentrations. (C,D) Boxplots (n = 6) present the frequencies of single IFN-γ, single TNF-α, single IL-17A (C) , IFN-γ + TNF-α + , and TNF-α + IL-17A + CD4 + T cells (D) . Different letters on boxplots indicate significant differences ( p
Figure Legend Snippet: Frequencies of IFN-γ, TNF-α, and IL-17A producing CD4 + T cells in the presence of DON and DOM-1. Violet proliferation dye-stained PBMCs were cultivated for 4 days in the presence of ConA alone or in combination with two different DON concentrations (0.2 and 0.8 μM) and DOM-1 (16 μM). Following PMA/Ionomycin stimulation for 4 h on the fourth day, the cells were harvested and analyzed for cytokine production. (A) Total CD4 + T cells were gated and analyzed for production of IFN-γ, TNF-α, and IL-17A. IFN-γ single producing T cells are designated with (1), single TNF-α with (2), total IFN-γ with (1 + 3), total TNF-α with (2 + 3), double IFN-γ + TNF-α + with (3), single IL-17A with (4), total IL-17A with (4 + 5), and double TNF-α + IL-17A + with (5). Representative flow cytometry data from one animal is shown. Red numbers indicate percentages of cytokine-producing CD4 + T-cell subsets. (B) Boxplots summarize the data of experiments with PBMCs from six pigs and show the frequencies of total IFN-γ, total TNF-α, and total IL-17A producing CD4 + T cells for the different DON and DOM-1 concentrations. (C,D) Boxplots (n = 6) present the frequencies of single IFN-γ, single TNF-α, single IL-17A (C) , IFN-γ + TNF-α + , and TNF-α + IL-17A + CD4 + T cells (D) . Different letters on boxplots indicate significant differences ( p

Techniques Used: Staining, Flow Cytometry

Frequencies of IFN-γ and TNF-α producing CD8 + T cells in the presence of DON and DOM-1. Violet proliferation dye-stained PBMCs were cultivated for 4 days in the presence of ConA alone or in combination with two different DON concentrations (0.2 and 0.8 μM) and DOM-1 (16 μM). Following PMA/Ionomycin stimulation for 4 h on the fourth day, the cells were harvested and analyzed for cytokine production. (A) Total CD8β + T cells were gated and analyzed for production of IFN-γ and TNF-α. IFN-γ single producing T cells are designated with (1), TNF-α single with (2), and IFN-γ + TNF-α + with (3). Representative flow cytometry data from one animal is shown. Red numbers indicate percentages of cytokine-producing CD8 + T-cell subsets. (B) The boxplots summarize the data of experiments with PBMCs from six individual animals and show the frequencies of total IFN-γ, total TNF-α, single IFN-γ, single TNF-α, and IFN-γ + TNF-α + producing CD8 + T cells. Different letters on boxplots indicate significant differences ( p
Figure Legend Snippet: Frequencies of IFN-γ and TNF-α producing CD8 + T cells in the presence of DON and DOM-1. Violet proliferation dye-stained PBMCs were cultivated for 4 days in the presence of ConA alone or in combination with two different DON concentrations (0.2 and 0.8 μM) and DOM-1 (16 μM). Following PMA/Ionomycin stimulation for 4 h on the fourth day, the cells were harvested and analyzed for cytokine production. (A) Total CD8β + T cells were gated and analyzed for production of IFN-γ and TNF-α. IFN-γ single producing T cells are designated with (1), TNF-α single with (2), and IFN-γ + TNF-α + with (3). Representative flow cytometry data from one animal is shown. Red numbers indicate percentages of cytokine-producing CD8 + T-cell subsets. (B) The boxplots summarize the data of experiments with PBMCs from six individual animals and show the frequencies of total IFN-γ, total TNF-α, single IFN-γ, single TNF-α, and IFN-γ + TNF-α + producing CD8 + T cells. Different letters on boxplots indicate significant differences ( p

Techniques Used: Staining, Flow Cytometry

Frequencies of IFN-γ and TNF-α producing cells in proliferating and non-proliferating CD8 + T cells in the presence of DON and DOM-1. Violet proliferation dye-stained PBMCs were cultivated for 4 days in the presence of ConA alone or in combination with two different DON concentrations (0.2 and 0.8 μM) and a range of DOM-1 (16 μM). Following PMA/Ionomycin stimulation for 4 h on the fourth day, the cells were harvested and analyzed for cytokine production. (A) Representative flow cytometry data showing the gating of CD8β + T cells and analysis of total IFN-γ and TNF-α producing cells within proliferating and non-proliferating CD8 T cells. Red numbers give percentages of cytokine producing cells. (B) Representative flow cytometry data showing the gating of proliferating (cells gated on the left) and non-proliferating (cells gated on the right) CD8 + T cells with subsequent analysis of IFN-γ single, TNF-α single, and IFN-γ/TNF-α co-producing CD8 + T cells in the presence of DON and DOM-1. Red numbers indicate percentages of cytokine-producing CD8 + T-cell subsets. (C) Boxplots summarize the data of experiments performed with PBMCs of six individual pigs and present the frequencies of proliferating and non-proliferating (parental) CD8 + T cells for the different cytokine producing subsets in the presence of DON and DOM-1. Different letters on boxplots indicate significant differences ( p
Figure Legend Snippet: Frequencies of IFN-γ and TNF-α producing cells in proliferating and non-proliferating CD8 + T cells in the presence of DON and DOM-1. Violet proliferation dye-stained PBMCs were cultivated for 4 days in the presence of ConA alone or in combination with two different DON concentrations (0.2 and 0.8 μM) and a range of DOM-1 (16 μM). Following PMA/Ionomycin stimulation for 4 h on the fourth day, the cells were harvested and analyzed for cytokine production. (A) Representative flow cytometry data showing the gating of CD8β + T cells and analysis of total IFN-γ and TNF-α producing cells within proliferating and non-proliferating CD8 T cells. Red numbers give percentages of cytokine producing cells. (B) Representative flow cytometry data showing the gating of proliferating (cells gated on the left) and non-proliferating (cells gated on the right) CD8 + T cells with subsequent analysis of IFN-γ single, TNF-α single, and IFN-γ/TNF-α co-producing CD8 + T cells in the presence of DON and DOM-1. Red numbers indicate percentages of cytokine-producing CD8 + T-cell subsets. (C) Boxplots summarize the data of experiments performed with PBMCs of six individual pigs and present the frequencies of proliferating and non-proliferating (parental) CD8 + T cells for the different cytokine producing subsets in the presence of DON and DOM-1. Different letters on boxplots indicate significant differences ( p

Techniques Used: Staining, Flow Cytometry

32) Product Images from "Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides"

Article Title: Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20061308

IL-22 transcript expression is high in Th17 cells. Purified CD62L hi CD4 + (naive) DO11 T cells were activated with irradiated splenocytes, OVA 323–339 (OVAp), and various cytokines and antibodies to differentiate cells to Th1 (IL-12, anti–IL-4), Th2 (IL-4, anti–IFN-γ), and Th17 (TGF-β, IL-6, IL-1β, TNF-α, IL-23, anti–IFN-γ, anti–IL-4) lineages. On day 7 of culture, CD4 + T cells were repurified and rested overnight. Cells were then restimulated with PMA and ionomycin under differentiation conditions for 6 h. RNA was prepared and quantitative PCR was performed in A to ensure that cells were properly differentiated. (B and C) ILs known to be expressed in T cells were evaluated by quantitative PCR. All transcript amounts were normalized to HPRT, and fold induction was calculated relative to unactivated, naive DO11 T cells. Data shown are representative of two independent experiments. Error bars are SD.
Figure Legend Snippet: IL-22 transcript expression is high in Th17 cells. Purified CD62L hi CD4 + (naive) DO11 T cells were activated with irradiated splenocytes, OVA 323–339 (OVAp), and various cytokines and antibodies to differentiate cells to Th1 (IL-12, anti–IL-4), Th2 (IL-4, anti–IFN-γ), and Th17 (TGF-β, IL-6, IL-1β, TNF-α, IL-23, anti–IFN-γ, anti–IL-4) lineages. On day 7 of culture, CD4 + T cells were repurified and rested overnight. Cells were then restimulated with PMA and ionomycin under differentiation conditions for 6 h. RNA was prepared and quantitative PCR was performed in A to ensure that cells were properly differentiated. (B and C) ILs known to be expressed in T cells were evaluated by quantitative PCR. All transcript amounts were normalized to HPRT, and fold induction was calculated relative to unactivated, naive DO11 T cells. Data shown are representative of two independent experiments. Error bars are SD.

Techniques Used: Expressing, Purification, Irradiation, Real-time Polymerase Chain Reaction

33) Product Images from "Myosin IIA is required for cytolytic granule exocytosis in human NK cells"

Article Title: Myosin IIA is required for cytolytic granule exocytosis in human NK cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20071143

Myosin II inhibitors block lytic granule exocytosis. (A) pNK and YTS cells were activated with either their respective target cells or PMA and ionomycin (Iono). The percentage of increase in CD107a surface expression was evaluated by FACS and expressed as the increase over identically treated (including with appropriate inhibitors) unstimulated cells. Thus, unstimulated cells were used as a baseline to account for any nonspecific stimulation of antibody endocytosis or granule exocytosis over the incubation time, as well as any antibody endocytosis nonspecifically induced by cell treatment. Unstimulated cells also accounted for any potential shifts in fluorescence caused by drug color. Bar graphs represent the mean ± the SD of three independent experiments. (B) A representative histogram of CD107a surface expression from pNK cells stimulated with PMA/ionomycin ± blebbistatin. The percentage of pNK cells expressing a high level of CD107a was gated, and values are shown on the graph. The percentage of Max indicates the number of events at each given fluorescence intensity, where the intensity with the greatest number of cells has been normalized to be 100%. (C) pNK cells were stimulated by plate-bound anti-CD16 or K562 target cells, and their supernatant was used in a serine esterase assay. Results are expressed as a percentage of total release from cells pretreated with inhibitors or appropriate controls to account for possible background effects on the colorimetric assay caused by either intrinsic compound color or direct effects on serine esterase activity. Bar graphs represent the mean ± the SD of four to five independent experiments. Paired and unpaired Student's t tests were performed to obtain P values.
Figure Legend Snippet: Myosin II inhibitors block lytic granule exocytosis. (A) pNK and YTS cells were activated with either their respective target cells or PMA and ionomycin (Iono). The percentage of increase in CD107a surface expression was evaluated by FACS and expressed as the increase over identically treated (including with appropriate inhibitors) unstimulated cells. Thus, unstimulated cells were used as a baseline to account for any nonspecific stimulation of antibody endocytosis or granule exocytosis over the incubation time, as well as any antibody endocytosis nonspecifically induced by cell treatment. Unstimulated cells also accounted for any potential shifts in fluorescence caused by drug color. Bar graphs represent the mean ± the SD of three independent experiments. (B) A representative histogram of CD107a surface expression from pNK cells stimulated with PMA/ionomycin ± blebbistatin. The percentage of pNK cells expressing a high level of CD107a was gated, and values are shown on the graph. The percentage of Max indicates the number of events at each given fluorescence intensity, where the intensity with the greatest number of cells has been normalized to be 100%. (C) pNK cells were stimulated by plate-bound anti-CD16 or K562 target cells, and their supernatant was used in a serine esterase assay. Results are expressed as a percentage of total release from cells pretreated with inhibitors or appropriate controls to account for possible background effects on the colorimetric assay caused by either intrinsic compound color or direct effects on serine esterase activity. Bar graphs represent the mean ± the SD of four to five independent experiments. Paired and unpaired Student's t tests were performed to obtain P values.

Techniques Used: Blocking Assay, Expressing, FACS, Incubation, Fluorescence, Esterase Assay, Colorimetric Assay, Activity Assay

34) Product Images from "Liver Is Able to Activate Na?ve CD8+ T Cells with Dysfunctional Anti-Viral Activity in the Murine System"

Article Title: Liver Is Able to Activate Na?ve CD8+ T Cells with Dysfunctional Anti-Viral Activity in the Murine System

Journal: PLoS ONE

doi: 10.1371/journal.pone.0007619

IV adenovirus infection causes a robust defect in CD8 + T cell effector function with elevated PD-1 expression in the liver. (A, B) 0.5×10 6 Thy1.1 + OT-1 + CD8 + T cells were adoptively transferred into naïve Thy1.2 + C57BL/6 mice. These mice were allowed to rest for one day and were then infected with 5×10 8 PFU Ad-OVA via either SubQ or IV injection (A) or 5×10 8 , 5×10 7 , 5×10 6 PFU Ad-OVA via IV injection (B). At day 7 p.i., liver leukocytes were isolated and then restimulated directly ex vivo with PMA/ionomycin (A) or OVA peptide in the presence of monensin (B) for 5 hours. The plots are gated on live cells and the numbers represent the percentage of cells within the indicated gates. (C) 2×10 6 Thy1.1 + OT-1 + CD8 + T cells were adoptively transferred into naïve Thy1.2 + C57BL/6 mice. These mice were allowed to rest for one day and then they were infected with 5×10 8 PFU Ad-OVA via either SubQ or IV injection. At day 7 p.i., liver leukocytes were isolated and the percentage of Thy1.1 + CD8 + T cells expressing PD-1 was determined. The expression of PD-1 by CD8 + T cells in the spleen, lung, and liver following 5×10 8 PFU Ad-OVA IV infection was also evaluated (right histogram). Both of the plots are gated on CD8 + T cells. Data are representative of two independent experiments ( n = 2/group). (D) Effect of PD-1 blockade on the liver primed CD8 + T cell effector activity. 2×10 6 CFSE labeled Thy1.1 + OT-1 + CD8 + T cells were adoptively transferred into Thy1.2 + C57BL/6 mice treated with anti-B7-H1 or control antibody (200 ug per mouse) one day prior to adoptive transfer. The recipient mice were infected with 5×10 8 PFU Ad-OVA via IV administration one day later. At 48 hours p.i., liver and spleen cells were isolated and then restimulated directly ex vivo with OVA peptide for 5 hours in the presence of monensin. The production of IFN-γ by proliferating Thy1.1 + CD8 + T cells was analyzed by flow cytometry and the plots are gated on Thy1.1 + CD8 + T cells.
Figure Legend Snippet: IV adenovirus infection causes a robust defect in CD8 + T cell effector function with elevated PD-1 expression in the liver. (A, B) 0.5×10 6 Thy1.1 + OT-1 + CD8 + T cells were adoptively transferred into naïve Thy1.2 + C57BL/6 mice. These mice were allowed to rest for one day and were then infected with 5×10 8 PFU Ad-OVA via either SubQ or IV injection (A) or 5×10 8 , 5×10 7 , 5×10 6 PFU Ad-OVA via IV injection (B). At day 7 p.i., liver leukocytes were isolated and then restimulated directly ex vivo with PMA/ionomycin (A) or OVA peptide in the presence of monensin (B) for 5 hours. The plots are gated on live cells and the numbers represent the percentage of cells within the indicated gates. (C) 2×10 6 Thy1.1 + OT-1 + CD8 + T cells were adoptively transferred into naïve Thy1.2 + C57BL/6 mice. These mice were allowed to rest for one day and then they were infected with 5×10 8 PFU Ad-OVA via either SubQ or IV injection. At day 7 p.i., liver leukocytes were isolated and the percentage of Thy1.1 + CD8 + T cells expressing PD-1 was determined. The expression of PD-1 by CD8 + T cells in the spleen, lung, and liver following 5×10 8 PFU Ad-OVA IV infection was also evaluated (right histogram). Both of the plots are gated on CD8 + T cells. Data are representative of two independent experiments ( n = 2/group). (D) Effect of PD-1 blockade on the liver primed CD8 + T cell effector activity. 2×10 6 CFSE labeled Thy1.1 + OT-1 + CD8 + T cells were adoptively transferred into Thy1.2 + C57BL/6 mice treated with anti-B7-H1 or control antibody (200 ug per mouse) one day prior to adoptive transfer. The recipient mice were infected with 5×10 8 PFU Ad-OVA via IV administration one day later. At 48 hours p.i., liver and spleen cells were isolated and then restimulated directly ex vivo with OVA peptide for 5 hours in the presence of monensin. The production of IFN-γ by proliferating Thy1.1 + CD8 + T cells was analyzed by flow cytometry and the plots are gated on Thy1.1 + CD8 + T cells.

Techniques Used: Infection, Expressing, Mouse Assay, IV Injection, Isolation, Ex Vivo, Activity Assay, Labeling, Adoptive Transfer Assay, Flow Cytometry, Cytometry

35) Product Images from "CTLA4 blockade increases Th17 cells in patients with metastatic melanoma"

Article Title: CTLA4 blockade increases Th17 cells in patients with metastatic melanoma

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-7-35

Increase in Th17 cells after tremelimumab-based therapy by intracellular cytokine staining . A ) Gating strategy for IL-17 intracellular staining. Starting from either whole PBMC or CD-4 sorted cells (as depicted here), the lymphocyte population was gated on by FSC-H and SSC-H dot plot. Live cells were gated in the same graphic. A second gate was performed in CD3 and SSC-H dot plot. We analyzed for IL-17-producing cells among CD4+ T cells after gating. B ) Example of IL-17 intracellular staining. After 4-day activation of CD4-sorted cells with anti-CD3 and anti-CD28, cells were additionally stimulated with PMA and ionomycin while inhibiting protein transport, and the number of Th17 cells was determined by flow cytometry. Depicted are the plots of gated Th17 cells from patient NRA12. The left column is the baseline pre-dosing sample, and the right column the post-dosing sample. C ) Th17 quantification by flow cytometry. Pre- and post-dosing whole PBMC (left graph) or CD4+ cells (right graph) analyzed by flow cytometry for Th17 cells as described above (p values by pairwise t-test).
Figure Legend Snippet: Increase in Th17 cells after tremelimumab-based therapy by intracellular cytokine staining . A ) Gating strategy for IL-17 intracellular staining. Starting from either whole PBMC or CD-4 sorted cells (as depicted here), the lymphocyte population was gated on by FSC-H and SSC-H dot plot. Live cells were gated in the same graphic. A second gate was performed in CD3 and SSC-H dot plot. We analyzed for IL-17-producing cells among CD4+ T cells after gating. B ) Example of IL-17 intracellular staining. After 4-day activation of CD4-sorted cells with anti-CD3 and anti-CD28, cells were additionally stimulated with PMA and ionomycin while inhibiting protein transport, and the number of Th17 cells was determined by flow cytometry. Depicted are the plots of gated Th17 cells from patient NRA12. The left column is the baseline pre-dosing sample, and the right column the post-dosing sample. C ) Th17 quantification by flow cytometry. Pre- and post-dosing whole PBMC (left graph) or CD4+ cells (right graph) analyzed by flow cytometry for Th17 cells as described above (p values by pairwise t-test).

Techniques Used: Staining, Activation Assay, Flow Cytometry, Cytometry

36) Product Images from "Therapeutic effect of the potent IL-12/IL-23 inhibitor STA-5326 on experimental autoimmune uveoretinitis"

Article Title: Therapeutic effect of the potent IL-12/IL-23 inhibitor STA-5326 on experimental autoimmune uveoretinitis

Journal: Arthritis Research & Therapy

doi: 10.1186/ar2530

Antigen specific proliferation is decreased in lymph node cells of STA-5326-treated mice, and IL-17 production and the proportion of Th17 cells from draining lymph nodes are significantly reduced in STA-5326-treated mice . (a) Antigen specific proliferation of draining lymph nodes in STA-5326-treated or vehicle-treated mice. Immunized mice were treated with 5 mg/kg or 20 mg/kg STA-5326 or vehicle from day 0 to day 14 after immunization. Draining lymph node cells collected on day 18 after immunization were pooled within each group. Cultures were stimulated with 10 μg/ml human experimental autoimmune uveoretinitis (IRBP) peptide 1–20 for 72 hours and pulsed with bromodeoxyuridine for the last 24 hours. (b-d) Cytokine production of interferon (IFN) γ, interleukin (IL) 17 and IL-4 by draining lymph node cells from STA-5326-treated or vehicle-treated mice. Immunized mice were treated with 5 mg/kg or 20 mg/kg STA-5326 or vehicle from day 0 to day 14 after immunization. Draining lymph node cells collected on day 18 after immunization were pooled within each group. Cultures were stimulated with 10 μg/ml IRBP 1–20 for 72 hours, and supernatants collected at 72 hours were assayed by ELISA. (a-d) Statistical analysis was performed using Student;s t -test. (e and f) Intracellular cytokine staining of draining lymph node cells in 20 mg/kg STA-5326 or vehicle-treated mice. Draining lymph node cells collected on day 18 were stimulated with IRBP 1–20 for 72 hours, and the cultured cells were incubated with PMA plus ionomycin and brefeldin A and stained with CD4, CD8 and intracellular IFN-γ and IL-17. The percentage shown in the upper right quadrant is for IFN-γ or IL-17 positive cells in CD4 + T cells.
Figure Legend Snippet: Antigen specific proliferation is decreased in lymph node cells of STA-5326-treated mice, and IL-17 production and the proportion of Th17 cells from draining lymph nodes are significantly reduced in STA-5326-treated mice . (a) Antigen specific proliferation of draining lymph nodes in STA-5326-treated or vehicle-treated mice. Immunized mice were treated with 5 mg/kg or 20 mg/kg STA-5326 or vehicle from day 0 to day 14 after immunization. Draining lymph node cells collected on day 18 after immunization were pooled within each group. Cultures were stimulated with 10 μg/ml human experimental autoimmune uveoretinitis (IRBP) peptide 1–20 for 72 hours and pulsed with bromodeoxyuridine for the last 24 hours. (b-d) Cytokine production of interferon (IFN) γ, interleukin (IL) 17 and IL-4 by draining lymph node cells from STA-5326-treated or vehicle-treated mice. Immunized mice were treated with 5 mg/kg or 20 mg/kg STA-5326 or vehicle from day 0 to day 14 after immunization. Draining lymph node cells collected on day 18 after immunization were pooled within each group. Cultures were stimulated with 10 μg/ml IRBP 1–20 for 72 hours, and supernatants collected at 72 hours were assayed by ELISA. (a-d) Statistical analysis was performed using Student;s t -test. (e and f) Intracellular cytokine staining of draining lymph node cells in 20 mg/kg STA-5326 or vehicle-treated mice. Draining lymph node cells collected on day 18 were stimulated with IRBP 1–20 for 72 hours, and the cultured cells were incubated with PMA plus ionomycin and brefeldin A and stained with CD4, CD8 and intracellular IFN-γ and IL-17. The percentage shown in the upper right quadrant is for IFN-γ or IL-17 positive cells in CD4 + T cells.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining, Cell Culture, Incubation

37) Product Images from "Thymic expression of IL-4 and IL-15 after systemic inflammatory or infectious Th1 disease processes induce the acquisition of "innate" characteristics during CD8+ T cell development"

Article Title: Thymic expression of IL-4 and IL-15 after systemic inflammatory or infectious Th1 disease processes induce the acquisition of "innate" characteristics during CD8+ T cell development

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007456

Blockage of IL-4 and IL-15 inhibits the induction of the innate phenotype in DP thymocytes. A bulk population of CD45.2 + thymocytes either from WT control, WT T . cruzi- infected (Tulahuen) or IL-4KO T . cruzi- infected (Tulahuen) mice were obtained at day 14 post-infection and cultured for 2h at 37°C in the presence of PMA/ionomycin. Cells were washed twice and co-cultured with sorted DP cells from OT-I (Vβ5 + OVA-tetramer + ) control mice at a 1:1 ratio in the presence or absence of a neutralizing anti-IL-15 Ab. After 48h, thymocytes were obtained and CD44, CD122, CD49d, Eomes and Tbet expression were analyzed by Flow cytometry only in DP CD8 OVA-specific OT-I thymocytes. Eomes or Tbet were measured by intranuclear staining using Flow cytometry analysis and were expressed as the difference of the mean fluorescence intensity (MFI) of Eomes or Tbet vs the MFI of the correspondent isotype control (IC). Histograms are representative of two independent experiments with 3–6 mice/experiment. The statistical test applied was a One-way ANOVA. T . cruzi vs the rest of the groups, *p
Figure Legend Snippet: Blockage of IL-4 and IL-15 inhibits the induction of the innate phenotype in DP thymocytes. A bulk population of CD45.2 + thymocytes either from WT control, WT T . cruzi- infected (Tulahuen) or IL-4KO T . cruzi- infected (Tulahuen) mice were obtained at day 14 post-infection and cultured for 2h at 37°C in the presence of PMA/ionomycin. Cells were washed twice and co-cultured with sorted DP cells from OT-I (Vβ5 + OVA-tetramer + ) control mice at a 1:1 ratio in the presence or absence of a neutralizing anti-IL-15 Ab. After 48h, thymocytes were obtained and CD44, CD122, CD49d, Eomes and Tbet expression were analyzed by Flow cytometry only in DP CD8 OVA-specific OT-I thymocytes. Eomes or Tbet were measured by intranuclear staining using Flow cytometry analysis and were expressed as the difference of the mean fluorescence intensity (MFI) of Eomes or Tbet vs the MFI of the correspondent isotype control (IC). Histograms are representative of two independent experiments with 3–6 mice/experiment. The statistical test applied was a One-way ANOVA. T . cruzi vs the rest of the groups, *p

Techniques Used: Infection, Mouse Assay, Cell Culture, Expressing, Flow Cytometry, Cytometry, Staining, Fluorescence

38) Product Images from "Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors"

Article Title: Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors

Journal: Nature Communications

doi: 10.1038/s41467-018-05072-0

Phenotypic and functional properties of DP, SP and DN CD8 TILs. a Ex vivo phenotypic analysis of the expression of surface proteins CD69, CCR7, CD127, and CD28, delineating a T RM phenotype, on the three CD8 TIL subsets (top, one representative patient shown, colors match key). Summary of the frequency/expression level of the above-mentioned markers among several cancer patients ( n = 12 for CD69; n = 10 for CCR7; n = 13 for CD127; and n = 6 for CD28, bottom). Each dot represents one patient. b Ex vivo phenotypic analysis of the expression of surface and intracellular proteins PD-1, CTLA-4, TIM-3, 4-1BB, and Ki-67, delineating an activated/chronically stimulated phenotype, on the three CD8 TIL subsets (top, one representative patient shown, colors match key). Summary of the frequency of the above-mentioned markers among several cancer patients ( n = 11 for PD-1; n = 5 for CTLA-4; n = 8 for TIM-3; n = 8 for 4-1BB; and n = 17 for Ki-67, bottom). Each dot represents one patient. c Representative flow cytometric analysis of IFN-γ and TNF-α production by the three CD8 TIL subsets stimulated for 4 h with Phorbol 12-Myristate 13-Acetate (PMA)/ionomycin. Numbers in each quadrant indicate percent cells positive for IFN-γ and/or TNF-α for each subset. d Frequency of IFN-γ, TNF-α, IFN-γ/TNF-α double-positive cells by each CD8 TIL subsets. Data are from 6 HNSCC patients. e Representative flow cytometric analysis of granzyme B production by CD8 TIL subsets (left, colors match key) and summary of six different HNSCC patients (right). Small horizontal lines indicate mean ± SEM; * p
Figure Legend Snippet: Phenotypic and functional properties of DP, SP and DN CD8 TILs. a Ex vivo phenotypic analysis of the expression of surface proteins CD69, CCR7, CD127, and CD28, delineating a T RM phenotype, on the three CD8 TIL subsets (top, one representative patient shown, colors match key). Summary of the frequency/expression level of the above-mentioned markers among several cancer patients ( n = 12 for CD69; n = 10 for CCR7; n = 13 for CD127; and n = 6 for CD28, bottom). Each dot represents one patient. b Ex vivo phenotypic analysis of the expression of surface and intracellular proteins PD-1, CTLA-4, TIM-3, 4-1BB, and Ki-67, delineating an activated/chronically stimulated phenotype, on the three CD8 TIL subsets (top, one representative patient shown, colors match key). Summary of the frequency of the above-mentioned markers among several cancer patients ( n = 11 for PD-1; n = 5 for CTLA-4; n = 8 for TIM-3; n = 8 for 4-1BB; and n = 17 for Ki-67, bottom). Each dot represents one patient. c Representative flow cytometric analysis of IFN-γ and TNF-α production by the three CD8 TIL subsets stimulated for 4 h with Phorbol 12-Myristate 13-Acetate (PMA)/ionomycin. Numbers in each quadrant indicate percent cells positive for IFN-γ and/or TNF-α for each subset. d Frequency of IFN-γ, TNF-α, IFN-γ/TNF-α double-positive cells by each CD8 TIL subsets. Data are from 6 HNSCC patients. e Representative flow cytometric analysis of granzyme B production by CD8 TIL subsets (left, colors match key) and summary of six different HNSCC patients (right). Small horizontal lines indicate mean ± SEM; * p

Techniques Used: Functional Assay, Ex Vivo, Expressing, Flow Cytometry

39) Product Images from "CXCR4 blockade decreases CD4+ T cell exhaustion and improves survival in a murine model of polymicrobial sepsis"

Article Title: CXCR4 blockade decreases CD4+ T cell exhaustion and improves survival in a murine model of polymicrobial sepsis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0188882

Cytokine production by CD4+ and CD8+ T cells is similar in plerixafor-treated septic animals as compared to control septic animals. Control or plerixafor-treated septic animals were sacrificed at 24h post-CLP and splenocytes were restimulated ex vivo with PMA/ionomycin for 4 h. Cells were fixed, permeabilized, and frequencies of IL-2 (A-B), IFN-g (C-D), and TNF (E-F) secreting CD4+ (A, C, E) and CD8+ (B, D, F) T cells were assessed by flow cytometry. Data shown are cumulative from two independent experiments (n = 4-8/group).
Figure Legend Snippet: Cytokine production by CD4+ and CD8+ T cells is similar in plerixafor-treated septic animals as compared to control septic animals. Control or plerixafor-treated septic animals were sacrificed at 24h post-CLP and splenocytes were restimulated ex vivo with PMA/ionomycin for 4 h. Cells were fixed, permeabilized, and frequencies of IL-2 (A-B), IFN-g (C-D), and TNF (E-F) secreting CD4+ (A, C, E) and CD8+ (B, D, F) T cells were assessed by flow cytometry. Data shown are cumulative from two independent experiments (n = 4-8/group).

Techniques Used: Ex Vivo, Flow Cytometry, Cytometry

40) Product Images from "mTOR coordinates transcriptional programs and mitochondrial metabolism of activated Treg subsets to protect tissue homeostasis"

Article Title: mTOR coordinates transcriptional programs and mitochondrial metabolism of activated Treg subsets to protect tissue homeostasis

Journal: Nature Communications

doi: 10.1038/s41467-018-04392-5

mTOR is essential for activated T reg cell function. a Enrichment plots of the Hallmark mTORC1 (left) and Hallmark PI3K-Akt-mTOR (right) signaling pathways in activated T reg (aT reg ) compared to resting T reg (rT reg ) cells, identified by gene set enrichment analysis (GSEA). The top 20 enriched genes in each pathway (position indicated by the vertical black line) are listed to the right of each plot. b In vitro suppressive activity of T reg cells activated in the presence or absence of PP242. T N : naive CD4 + T cells. c Flow cytometry analysis of CTLA4 expression in T reg cells activated in the presence or absence of PP242. d In vitro suppressive activity of T reg cells isolated from Cd4 Cre Mtor +/+ or +/fl or Cd4 Cre Mtor fl/fl mice. e Representative image of lymphadenopathy in Foxp3 Cre/DTR Mtor fl/fl mice after DT treatment (left). Right, cell numbers of the spleen and peripheral lymph nodes (pLN) of Foxp3 Cre/DTR Mtor +/+ or +/fl or Foxp3 Cre/DTR Mtor fl/fl mice. f Flow cytometry analysis of naive and effector/memory CD4 + Foxp3-YFP − in pLN. g , h Cells from the spleen and pLN of Foxp3 Cre/DTR Mtor +/+ or +/fl or Foxp3 Cre/DTR Mtor fl/fl mice that received DT treatments were stimulated using PMA and ionomycin for 4–5 h. g Flow cytometry analysis of IL-4-producing CD4 + T cells in the spleen and pLN. h Quantification of IFN-γ + CD4 + Foxp3 − and CD8 + T cells or IL-4 + and IL-17A + CD4 + Foxp3 − T cells in the spleen. Error bars show mean ± s.e.m. * P
Figure Legend Snippet: mTOR is essential for activated T reg cell function. a Enrichment plots of the Hallmark mTORC1 (left) and Hallmark PI3K-Akt-mTOR (right) signaling pathways in activated T reg (aT reg ) compared to resting T reg (rT reg ) cells, identified by gene set enrichment analysis (GSEA). The top 20 enriched genes in each pathway (position indicated by the vertical black line) are listed to the right of each plot. b In vitro suppressive activity of T reg cells activated in the presence or absence of PP242. T N : naive CD4 + T cells. c Flow cytometry analysis of CTLA4 expression in T reg cells activated in the presence or absence of PP242. d In vitro suppressive activity of T reg cells isolated from Cd4 Cre Mtor +/+ or +/fl or Cd4 Cre Mtor fl/fl mice. e Representative image of lymphadenopathy in Foxp3 Cre/DTR Mtor fl/fl mice after DT treatment (left). Right, cell numbers of the spleen and peripheral lymph nodes (pLN) of Foxp3 Cre/DTR Mtor +/+ or +/fl or Foxp3 Cre/DTR Mtor fl/fl mice. f Flow cytometry analysis of naive and effector/memory CD4 + Foxp3-YFP − in pLN. g , h Cells from the spleen and pLN of Foxp3 Cre/DTR Mtor +/+ or +/fl or Foxp3 Cre/DTR Mtor fl/fl mice that received DT treatments were stimulated using PMA and ionomycin for 4–5 h. g Flow cytometry analysis of IL-4-producing CD4 + T cells in the spleen and pLN. h Quantification of IFN-γ + CD4 + Foxp3 − and CD8 + T cells or IL-4 + and IL-17A + CD4 + Foxp3 − T cells in the spleen. Error bars show mean ± s.e.m. * P

Techniques Used: Cell Function Assay, In Vitro, Activity Assay, Flow Cytometry, Cytometry, Expressing, Isolation, Mouse Assay

Disruption of mTOR in T reg cells results in fatal autoimmunity. a Representative image of 47-day-old Foxp3 Cre Mtor +/fl and Foxp3 Cre Mtor fl/fl littermates. b Representative image of lymphadenopathy in 47-day-old Foxp3 Cre Mtor fl/fl mice (left). Right, cell numbers of the spleen and peripheral lymph nodes (pLN) of Foxp3 Cre Mtor +/+ , Foxp3 Cre Mtor +/fl , or Foxp3 Cre Mtor fl/fl mice. The numbers of mice per group are indicated. c Representative hematoxylin and eosin staining of the indicated tissues from 6-week-old Foxp3 Cre Mtor +/fl and Foxp3 Cre Mtor fl/fl mice. The magnifications are indicated above the respective images for each tissue. d Survival curve of Foxp3 Cre Mtor +/fl and Foxp3 Cre Mtor fl/fl mice. The numbers of mice per group are indicated. e Flow cytometry analysis of naive and effector/memory CD4 + Foxp3-YFP – (depicted as CD4 + ) or CD8 + T-cell populations. f Splenocytes from Foxp3 Cre Mtor +/+ or +/fl and Foxp3 Cre Mtor fl/fl mice were stimulated using PMA and ionomycin for 4–5 h. Cytokine production by CD4 + and CD8 + T cells was assessed by flow cytometry and quantified. g Flow cytometry analysis of PD-1 + CXCR5 + T FH cells. Right, frequency and number of T FH cells in Foxp3 Cre Mtor +/+ or +/fl and Foxp3 Cre Mtor fl/fl mice. h Flow cytometry analysis of CD95 + GL7 + GC B cells. Right, frequency and number of GC B cells in Foxp3 Cre Mtor +/+ or +/fl and Foxp3 Cre Mtor fl/fl mice. Error bars show mean ± s.e.m. * P
Figure Legend Snippet: Disruption of mTOR in T reg cells results in fatal autoimmunity. a Representative image of 47-day-old Foxp3 Cre Mtor +/fl and Foxp3 Cre Mtor fl/fl littermates. b Representative image of lymphadenopathy in 47-day-old Foxp3 Cre Mtor fl/fl mice (left). Right, cell numbers of the spleen and peripheral lymph nodes (pLN) of Foxp3 Cre Mtor +/+ , Foxp3 Cre Mtor +/fl , or Foxp3 Cre Mtor fl/fl mice. The numbers of mice per group are indicated. c Representative hematoxylin and eosin staining of the indicated tissues from 6-week-old Foxp3 Cre Mtor +/fl and Foxp3 Cre Mtor fl/fl mice. The magnifications are indicated above the respective images for each tissue. d Survival curve of Foxp3 Cre Mtor +/fl and Foxp3 Cre Mtor fl/fl mice. The numbers of mice per group are indicated. e Flow cytometry analysis of naive and effector/memory CD4 + Foxp3-YFP – (depicted as CD4 + ) or CD8 + T-cell populations. f Splenocytes from Foxp3 Cre Mtor +/+ or +/fl and Foxp3 Cre Mtor fl/fl mice were stimulated using PMA and ionomycin for 4–5 h. Cytokine production by CD4 + and CD8 + T cells was assessed by flow cytometry and quantified. g Flow cytometry analysis of PD-1 + CXCR5 + T FH cells. Right, frequency and number of T FH cells in Foxp3 Cre Mtor +/+ or +/fl and Foxp3 Cre Mtor fl/fl mice. h Flow cytometry analysis of CD95 + GL7 + GC B cells. Right, frequency and number of GC B cells in Foxp3 Cre Mtor +/+ or +/fl and Foxp3 Cre Mtor fl/fl mice. Error bars show mean ± s.e.m. * P

Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry

Related Articles

Isolation:

Article Title: Phenotypic Analysis of Prostate-Infiltrating Lymphocytes Reveals TH17 and Treg Skewing
Article Snippet: .. Isolated CD4+ T cells from prostate and peripheral blood were resuspended in CTL medium with 0.05 µg/mL of phorbol 12-myristate 13-acetate, 0.5 µg/mL of ionomycin, and 1:1,000 GolgiStop (BD Biosciences) and plated at a density of < 1 × 106 in 96-well U-bottomed plates. .. Surface staining with a PE-Cy5 labeled mAb to CD45RO (BD Biosciences) was done prior to cell permeabilization.

Purification:

Article Title: Chemokine Receptor Expression Identifies Pre-T Helper (Th)1, Pre-Th2, and Nonpolarized Cells among Human CD4+ Central Memory T Cells
Article Snippet: .. Cytokine-producing capacity of FACS-purified subsets was assessed after stimulation of purified cell populations at 5 × 104 /100 μl for 24 h with 50 nM phorbol-12-13-dibutyrate (PdBu) and 0.5 μg/ml ionomycin, or in wells coated with 2 μg/ml each of anti-CD3 (clone TR66) and anti-CD28 antibodies (BD Biosciences). .. Cytokine concentrations of supernatants were then assessed by ELISA according to a standard protocol and analyzed with the Softmax program.

Incubation:

Article Title: CD11c+ monocyte/macrophages promote chronic Helicobacter hepaticus-induced intestinal inflammation through the production of IL-23
Article Snippet: .. For intracellular cytokine staining of T cells, cells were incubated for 3.5 h in complete IMDM medium containing 0.1 μM phorbol 12-myristate 13-acetate and 1 μM ionomycin with 1:1,000 GolgiPlug and GolgiStop solutions (BD Biosciences) at 37 °C in a humidified incubator with 5% CO2 . .. Following surface staining, cells were fixed and permeabilized with the Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) according to the manufacturer's instructions.

FACS:

Article Title: Chemokine Receptor Expression Identifies Pre-T Helper (Th)1, Pre-Th2, and Nonpolarized Cells among Human CD4+ Central Memory T Cells
Article Snippet: .. Cytokine-producing capacity of FACS-purified subsets was assessed after stimulation of purified cell populations at 5 × 104 /100 μl for 24 h with 50 nM phorbol-12-13-dibutyrate (PdBu) and 0.5 μg/ml ionomycin, or in wells coated with 2 μg/ml each of anti-CD3 (clone TR66) and anti-CD28 antibodies (BD Biosciences). .. Cytokine concentrations of supernatants were then assessed by ELISA according to a standard protocol and analyzed with the Softmax program.

Staining:

Article Title: CXCL4 is a novel inducer of human Th17 cells and correlates with IL‐17 and IL‐22 in psoriatic arthritis
Article Snippet: .. For intracellular cytokine staining, PMA, ionomycin and GolgiStop (#554724, BD Biosciences) was added for the final four hours of culture. .. For proliferation analysis, CD4+ T cells were labeled with CellTrace Violet (1.5 μM; #C34557 Thermo Fisher Scientific) prior culture.

Article Title: CD11c+ monocyte/macrophages promote chronic Helicobacter hepaticus-induced intestinal inflammation through the production of IL-23
Article Snippet: .. For intracellular cytokine staining of T cells, cells were incubated for 3.5 h in complete IMDM medium containing 0.1 μM phorbol 12-myristate 13-acetate and 1 μM ionomycin with 1:1,000 GolgiPlug and GolgiStop solutions (BD Biosciences) at 37 °C in a humidified incubator with 5% CO2 . .. Following surface staining, cells were fixed and permeabilized with the Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) according to the manufacturer's instructions.

CTL Assay:

Article Title: Phenotypic Analysis of Prostate-Infiltrating Lymphocytes Reveals TH17 and Treg Skewing
Article Snippet: .. Isolated CD4+ T cells from prostate and peripheral blood were resuspended in CTL medium with 0.05 µg/mL of phorbol 12-myristate 13-acetate, 0.5 µg/mL of ionomycin, and 1:1,000 GolgiStop (BD Biosciences) and plated at a density of < 1 × 106 in 96-well U-bottomed plates. .. Surface staining with a PE-Cy5 labeled mAb to CD45RO (BD Biosciences) was done prior to cell permeabilization.

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    Becton Dickinson ionomycin
    PKM2 Tetramerization Blocks T Cell Activation In Vitro Murine CD4 + CD62 + T cells were stimulated in vitro with CD3/CD28 antibodies in the presence of DMSO (CTRL), TEPP-46 50 μM, or 100 μM. (A and B) Cells were collected after 24 h of stimulation. (A) Quantification of Il2 mRNA in activated T cells by qRT-PCR (n = 9 from three independent experiments). (B) Cells were re-stimulated in vitro with PMA and <t>ionomycin</t> in the presence of brefeldin A. IL-2 production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced IL-2 production by TEPP-46-treated cells. Right, quantification of the percentage of IL-2-producing cells and IL-2 mean fluorescence intensity (MFI) in CTRL versus TEPP-46-treated cells (n = 8 from 3 independent experiments). (C–F) Cells were collected after 3 days of stimulation. (C) Top, representative flow cytometry plot displaying T cell proliferation assessed as CellTrace violet dilution. Bottom, a division index was calculated with FlowJo software to quantify T cell proliferation (n = 5 from four independent experiments). (D) Expression of surface CD62L, CD44, and CD25 was evaluated by flow cytometry. The percentage of expressing cells and the MFI are shown (n = 3 from 2 independent experiments). (E) Quantification of Tnfa mRNA levels in activated T cells by qRT-PCR (n = 6 from 6 independent experiments). (F) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. TNF-α production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of TNF-α-producing cells and TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 5 from 2 independent experiments). For all panels, data are the mean ± SD. ∗ p
    Ionomycin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1326 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PKM2 Tetramerization Blocks T Cell Activation In Vitro Murine CD4 + CD62 + T cells were stimulated in vitro with CD3/CD28 antibodies in the presence of DMSO (CTRL), TEPP-46 50 μM, or 100 μM. (A and B) Cells were collected after 24 h of stimulation. (A) Quantification of Il2 mRNA in activated T cells by qRT-PCR (n = 9 from three independent experiments). (B) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. IL-2 production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced IL-2 production by TEPP-46-treated cells. Right, quantification of the percentage of IL-2-producing cells and IL-2 mean fluorescence intensity (MFI) in CTRL versus TEPP-46-treated cells (n = 8 from 3 independent experiments). (C–F) Cells were collected after 3 days of stimulation. (C) Top, representative flow cytometry plot displaying T cell proliferation assessed as CellTrace violet dilution. Bottom, a division index was calculated with FlowJo software to quantify T cell proliferation (n = 5 from four independent experiments). (D) Expression of surface CD62L, CD44, and CD25 was evaluated by flow cytometry. The percentage of expressing cells and the MFI are shown (n = 3 from 2 independent experiments). (E) Quantification of Tnfa mRNA levels in activated T cells by qRT-PCR (n = 6 from 6 independent experiments). (F) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. TNF-α production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of TNF-α-producing cells and TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 5 from 2 independent experiments). For all panels, data are the mean ± SD. ∗ p

    Journal: Cell Metabolism

    Article Title: Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4+ T Cell Pathogenicity and Suppresses Autoimmunity

    doi: 10.1016/j.cmet.2019.10.015

    Figure Lengend Snippet: PKM2 Tetramerization Blocks T Cell Activation In Vitro Murine CD4 + CD62 + T cells were stimulated in vitro with CD3/CD28 antibodies in the presence of DMSO (CTRL), TEPP-46 50 μM, or 100 μM. (A and B) Cells were collected after 24 h of stimulation. (A) Quantification of Il2 mRNA in activated T cells by qRT-PCR (n = 9 from three independent experiments). (B) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. IL-2 production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced IL-2 production by TEPP-46-treated cells. Right, quantification of the percentage of IL-2-producing cells and IL-2 mean fluorescence intensity (MFI) in CTRL versus TEPP-46-treated cells (n = 8 from 3 independent experiments). (C–F) Cells were collected after 3 days of stimulation. (C) Top, representative flow cytometry plot displaying T cell proliferation assessed as CellTrace violet dilution. Bottom, a division index was calculated with FlowJo software to quantify T cell proliferation (n = 5 from four independent experiments). (D) Expression of surface CD62L, CD44, and CD25 was evaluated by flow cytometry. The percentage of expressing cells and the MFI are shown (n = 3 from 2 independent experiments). (E) Quantification of Tnfa mRNA levels in activated T cells by qRT-PCR (n = 6 from 6 independent experiments). (F) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. TNF-α production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced TNF-α production by TEPP-46 treated cells. Right, quantification of the percentage of TNF-α-producing cells and TNF-α MFI in CTRL versus TEPP-46-treated cells (n = 5 from 2 independent experiments). For all panels, data are the mean ± SD. ∗ p

    Article Snippet: CNS tissue homogenates were then re-suspended in 8 ml 40% isotonic Percoll before centrifugation at 1600 rpm for 20 min. Myelin debris was carefully removed, and CNS mononuclear cells were then passed through a 70 μm filter, washed, re-suspended in RPMI and stimulated with PMA (10 ng/ml), ionomycin (1 μg/ml), monensin (BD Golgistop, 1:1000; BD Biosciences) and brefeldin A (2 μg/ml) for 2 hours at 37°C.

    Techniques: Activation Assay, In Vitro, Quantitative RT-PCR, Flow Cytometry, Staining, Fluorescence, Software, Expressing

    Production of cytokines by Tconv and Treg cells during EAE. (A) Representative flow plots showing IL-17A and IFNγ production by CNS Tconv cells. (B) Frequencies of single and double cytokine producing Tconv cells in various organs at peak EAE upon stimulation with PMA and ionomycin. Data are from at least 5 independent experiments. (C) Percent of Tregs producing IL-10 or IFNγ in the various organs in response to PMA/Ionomycin stimulation. Data are from three independent experiments, except for SPL which is from two experiments.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Regulatory and T effector cells have overlapping low to high ranges in TCR affinities for self during demyelinating disease

    doi: 10.4049/jimmunol.1501464

    Figure Lengend Snippet: Production of cytokines by Tconv and Treg cells during EAE. (A) Representative flow plots showing IL-17A and IFNγ production by CNS Tconv cells. (B) Frequencies of single and double cytokine producing Tconv cells in various organs at peak EAE upon stimulation with PMA and ionomycin. Data are from at least 5 independent experiments. (C) Percent of Tregs producing IL-10 or IFNγ in the various organs in response to PMA/Ionomycin stimulation. Data are from three independent experiments, except for SPL which is from two experiments.

    Article Snippet: For intracellular cytokine staining, cells were stimulated with PMA and ionomycin for 4 hours in the presence of Brefeldin A, as described previously and then after staining surface markers, the cells were fixed and permeablized using the BD Cytofix/Cytoperm kit (BD Biosciences) followed by intracellular staining for cytokines ( ).

    Techniques: Flow Cytometry

    Eosinophils are significant producers of IL-6 in Ndfip1 −/− mice (A) The amount of IL-6 in the supernatants of splenocytes restimulated for 4.5–5 hours with PMA and ionomycin was measured by ELISA. Bar graph represents the mean + s.d. of triplicate samples from one mouse for each genotype. The results are representative of 3–4 mice per genotype. *p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The E3 ubiquitin ligase adaptor Ndfip1 regulates TH17 differentiation by limiting the production of pro-inflammatory cytokines 1

    doi: 10.4049/jimmunol.1102779

    Figure Lengend Snippet: Eosinophils are significant producers of IL-6 in Ndfip1 −/− mice (A) The amount of IL-6 in the supernatants of splenocytes restimulated for 4.5–5 hours with PMA and ionomycin was measured by ELISA. Bar graph represents the mean + s.d. of triplicate samples from one mouse for each genotype. The results are representative of 3–4 mice per genotype. *p

    Article Snippet: The cells were then washed and either cultured with PMA, ionomycin, and Golgi-stop (BD Bioseciences) for intracellular cytokine staining, or directly prepared for flow cytometry.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Reduced in vivo Th1 and Th17 responses in imiquimod-treated Padi2 –/– and Padi4 –/– mice. ( A and B ) Splenocytes from untreated and imiquimod-treated (IMQ-treated) FVB, Padi2 –/– , and Padi4 –/– mice were stimulated in vitro with PMA and ionomycin, and CD4 + T cell cytokine production was measured by flow cytometry to determine Th1 ( A ) and Th17 ( B ) responses. Box-and-whisker plots show median, lower and upper quartiles, and minimum and maximum % values and are representative of 2 independent experiments, each performed in 4-6 mice/group. * P

    Journal: JCI Insight

    Article Title: Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7–dependent lupus

    doi: 10.1172/jci.insight.124729

    Figure Lengend Snippet: Reduced in vivo Th1 and Th17 responses in imiquimod-treated Padi2 –/– and Padi4 –/– mice. ( A and B ) Splenocytes from untreated and imiquimod-treated (IMQ-treated) FVB, Padi2 –/– , and Padi4 –/– mice were stimulated in vitro with PMA and ionomycin, and CD4 + T cell cytokine production was measured by flow cytometry to determine Th1 ( A ) and Th17 ( B ) responses. Box-and-whisker plots show median, lower and upper quartiles, and minimum and maximum % values and are representative of 2 independent experiments, each performed in 4-6 mice/group. * P

    Article Snippet: For intracellular cytokine staining, cells were restimulated for 4 hours with PMA (50 ng/ml) and ionomycin (750 μg/ml), with the addition of Monensin (BD GolgiStop).

    Techniques: In Vivo, Mouse Assay, In Vitro, Flow Cytometry, Cytometry, Whisker Assay